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1.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-34470819

RESUMEN

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfolípidos/metabolismo , Membrana Celular/metabolismo , Polaridad Celular , Citoplasma/metabolismo , Exocitosis , Proteómica/métodos
2.
Plant J ; 110(5): 1382-1396, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35306706

RESUMEN

The exocyst complex is an octameric evolutionarily conserved tethering complex engaged in the regulation of polarized secretion in eukaryotic cells. Here, we focus on the systematic comparison of two isoforms of the SEC15 exocyst subunit, SEC15a and SEC15b. We infer that SEC15 gene duplication and diversification occurred in the common ancestor of seed plants (Spermatophytes). In Arabidopsis, SEC15a represents the main SEC15 isoform in the male gametophyte, and localizes to the pollen tube tip at the plasma membrane. Although pollen tubes of sec15a mutants are impaired, sporophytes show no phenotypic deviations. Conversely, SEC15b is the dominant isoform in the sporophyte and localizes to the plasma membrane in root and leaf cells. Loss-of-function sec15b mutants exhibit retarded elongation of hypocotyls and root hairs, a loss of apical dominance, dwarfed plant stature and reduced seed coat mucilage formation. Surprisingly, the sec15b mutants also exhibit compromised pollen tube elongation in vitro, despite its very low expression in pollen, suggesting a non-redundant role for the SEC15b isoform there. In pollen tubes, SEC15b localizes to distinct cytoplasmic structures. Reciprocally to this, SEC15a also functions in the sporophyte, where it accumulates at plasmodesmata. Importantly, although overexpressed SEC15a could fully complement the sec15b phenotypic deviations in the sporophyte, the pollen-specific overexpression of SEC15b was unable to fully compensate for the loss of SEC15a function in pollen. We conclude that the SEC15a and SEC15b isoforms evolved in seed plants, with SEC15a functioning mostly in pollen and SEC15b functioning mostly in the sporophyte.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Polen/metabolismo , Tubo Polínico/genética , Tubo Polínico/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Semillas/genética , Semillas/metabolismo
3.
Environ Microbiol ; 23(10): 6223-6240, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34472197

RESUMEN

Although many endophytic plant growth-promoting rhizobacteria have been identified, relatively little is still known about the mechanisms by which they enter plants and promote plant growth. The beneficial endophyte Enterobacter sp. SA187 was shown to maintain the productivity of crops in extreme agricultural conditions. Here we present that roots of its natural host (Indigofera argentea), alfalfa, tomato, wheat, barley and Arabidopsis are all efficiently colonized by SA187. Detailed analysis of the colonization process in Arabidopsis showed that colonization already starts during seed germination, where seed-coat mucilage supports SA187 proliferation. The meristematic zone of growing roots attracts SA187, allowing epiphytic colonization in the elongation zone. Unlike primary roots, lateral roots are significantly less epiphytically colonized by SA187. Root endophytic colonization was found to occur by passive entry of SA187 at lateral-root bases. However, SA187 also actively penetrates the root epidermis by enzymatic disruption of plant cell wall material. In contrast to roots, endophytic colonization of shoots occurs via stomata, whereby SA187 can actively re-open stomata similarly to pathogenic bacteria. In summary, several entry strategies were identified that allow SA187 to establish itself as a beneficial endophyte in several plant species, supporting its use as a plant growth-promoting bacterium in agriculture systems.


Asunto(s)
Arabidopsis , Enterobacter , Arabidopsis/microbiología , Productos Agrícolas , Endófitos/genética , Enterobacter/genética , Raíces de Plantas/microbiología
4.
Plant Physiol ; 184(4): 1823-1839, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33051268

RESUMEN

Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction.


Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Exocitosis/genética , Exocitosis/fisiología , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , Filogenia
5.
PLoS Genet ; 14(3): e1007273, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29554117

RESUMEN

Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that Enterobacter sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant Arabidopsis thaliana in vitro, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance.


Asunto(s)
Adaptación Fisiológica , Arabidopsis/fisiología , Enterobacter/fisiología , Etilenos/metabolismo , Metionina/análogos & derivados , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Regulación de la Expresión Génica de las Plantas , Metionina/biosíntesis , Metionina/metabolismo , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Potasio/metabolismo
6.
Proc Natl Acad Sci U S A ; 113(1): E41-50, 2016 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-26607451

RESUMEN

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endosomas/metabolismo , Exocitosis , Limoninas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Secuencia Conservada , Evolución Molecular , Humanos , Estructura Secundaria de Proteína
7.
Plant Physiol ; 174(1): 223-240, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28356503

RESUMEN

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollen-specific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Tubo Polínico/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Mutación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polen/genética , Polen/crecimiento & desarrollo , Polen/metabolismo , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Transporte Vesicular/metabolismo
8.
New Phytol ; 213(3): 1052-1067, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27801942

RESUMEN

Cortical microtubules (MTs) play a major role in the patterning of secondary cell wall (SCW) thickenings in tracheary elements (TEs) by determining the sites of SCW deposition. The EXO70A1 subunit of the exocyst secretory vesicle tethering complex was implicated to be important for TE development via the MT interaction. We investigated the subcellular localization of several exocyst subunits in the xylem of Arabidopsis thaliana and analyzed the functional significance of exocyst-mediated trafficking in TE development. Live cell imaging of fluorescently tagged exocyst subunits in TE using confocal microscopy and protein-protein interaction assays were performed to describe the role of the exocyst and its partners in TE development. In TEs, exocyst subunits were localized to the sites of SCW deposition in an MT-dependent manner. We propose that the mechanism of exocyst targeting to MTs involves the direct interaction of exocyst subunits with the COG2 protein. We demonstrated the importance of a functional exocyst subunit EXO84b for normal TE development and showed that the deposition of SCW constituents is partially compromised, possibly as a result of the mislocalization of secondary cellulose synthase in exocyst mutants. We conclude that the exocyst complex is an important factor bridging the pattern defined by cortical MTs with localized secretion of the SCW in developing TEs.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Microtúbulos/metabolismo , Xilema/crecimiento & desarrollo , Xilema/metabolismo , Arabidopsis/ultraestructura , Diferenciación Celular , Membrana Celular/metabolismo , Pared Celular/metabolismo , Secuencia Conservada , Glucosiltransferasas/metabolismo , Microtúbulos/ultraestructura , Modelos Biológicos , Mutación/genética , Haz Vascular de Plantas/metabolismo , Subunidades de Proteína/metabolismo , Xilema/citología , Xilema/ultraestructura
9.
Plant J ; 73(5): 709-19, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23163883

RESUMEN

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brefeldino A/farmacología , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Tipificación del Cuerpo , Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Mutación , Fenotipo , Epidermis de la Planta/citología , Epidermis de la Planta/efectos de los fármacos , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/citología , Plantones/efectos de los fármacos , Plantones/genética , Plantones/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Red trans-Golgi/metabolismo
10.
Plant Cell ; 22(9): 3053-65, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20870962

RESUMEN

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein-tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Citocinesis , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Mutagénesis Insercional , Mutación , Proteómica , Proteínas de Transporte Vesicular/genética
11.
Plant J ; 62(4): 615-27, 2010 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-20180921

RESUMEN

RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Gravitropismo , Brotes de la Planta/crecimiento & desarrollo , Transferasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brotes de la Planta/genética , Prenilación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transferasas/genética
12.
Front Plant Sci ; 11: 960, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32676093

RESUMEN

The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

13.
Plant Sci ; 280: 228-240, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30824001

RESUMEN

Salinity severely hampers crop productivity worldwide and plant growth promoting bacteria could serve as a sustainable solution to improve plant growth under salt stress. However, the molecular mechanisms underlying salt stress tolerance promotion by beneficial bacteria remain unclear. In this work, six bacterial isolates from four different desert plant species were screened for their biochemical plant growth promoting traits and salinity stress tolerance promotion of the unknown host plant Arabidopsis thaliana. Five of the isolates induced variable root phenotypes but could all increase plant shoot and root weight under salinity stress. Inoculation of Arabidopsis with five isolates under salinity stress resulted in tissue-specific transcriptional changes of ion transporters and reduced Na+/K+ shoot ratios. The work provides first insights into the possible mechanisms and the commonality by which phylogenetically diverse bacteria from different desert plants induce salinity stress tolerance in Arabidopsis. The bacterial isolates provide new tools for studying abiotic stress tolerance mechanisms in plants and a promising agricultural solution for increasing crop yields in semi-arid regions.


Asunto(s)
Arabidopsis/microbiología , Bacterias/clasificación , Fenómenos Fisiológicos Bacterianos , Regulación de la Expresión Génica de las Plantas , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bacterias/genética , Bacterias/aislamiento & purificación , Clima Desértico , Endófitos , Transporte Iónico , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Filogenia , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Raíces de Plantas/fisiología , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/microbiología , Brotes de la Planta/fisiología , Potasio/análisis , Estrés Salino , Tolerancia a la Sal , Sodio/análisis
14.
Front Plant Sci ; 4: 543, 2014 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-24427163

RESUMEN

Delivery and final fusion of the secretory vesicles with the relevant target membrane are hierarchically organized and reciprocally interconnected multi-step processes involving not only specific protein-protein interactions, but also specific protein-phospholipid interactions. The exocyst was discovered as a tethering complex mediating initial encounter of arriving exocytic vesicles with the plasma membrane. The exocyst complex is regulated by Rab and Rho small GTPases, resulting in docking of exocytic vesicles to the plasma membrane (PM) and finally their fusion mediated by specific SNARE complexes. In model Opisthokont cells, the exocyst was shown to directly interact with both microtubule and microfilament cytoskeleton and related motor proteins as well as with the PM via phosphatidylinositol 4, 5-bisphosphate specific binding, which directly affects cortical cytoskeleton and PM dynamics. Here we summarize the current knowledge on exocyst-cytoskeleton-PM interactions in order to open a perspective for future research in this area in plant cells.

15.
PLoS One ; 9(4): e94077, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24728280

RESUMEN

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.


Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Duplicación de Gen/genética , ADN Bacteriano/genética , Mutagénesis Insercional/genética
16.
Dev Cell ; 29(5): 607-620, 2014 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-24882377

RESUMEN

Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Citocinesis/fisiología , Placa de Crecimiento/citología , Proteínas de Transporte Vesicular/metabolismo , Arabidopsis/citología , Vesículas Citoplasmáticas/metabolismo , Exocitosis/fisiología , Placa de Crecimiento/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares
17.
Mol Biol Cell ; 24(4): 510-20, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23283982

RESUMEN

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Epidermis de la Planta/metabolismo , Raíces de Plantas/metabolismo , Proteínas SNARE/metabolismo , Arabidopsis/genética , Arabidopsis/ultraestructura , Proteínas de Arabidopsis/genética , Membrana Celular/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Exocitosis , Expresión Génica , Microscopía Fluorescente , Epidermis de la Planta/genética , Epidermis de la Planta/ultraestructura , Raíces de Plantas/genética , Raíces de Plantas/ultraestructura , Unión Proteica , Transporte de Proteínas , Proteínas SNARE/genética , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
18.
Plant Cell ; 20(5): 1330-45, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18492870

RESUMEN

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/fisiología , Proteínas de Transporte Vesicular/fisiología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Exocitosis/genética , Exocitosis/fisiología , Técnica del Anticuerpo Fluorescente Indirecta , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/genética , Polen/crecimiento & desarrollo , Polen/metabolismo , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Unión Proteica , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
19.
Plant J ; 48(1): 54-72, 2006 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16942608

RESUMEN

The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.


Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/crecimiento & desarrollo , Familia de Multigenes , Subunidades de Proteína/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Flores/anatomía & histología , Flores/crecimiento & desarrollo , Flores/metabolismo , Homocigoto , Hipocótilo/anatomía & histología , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Mutagénesis Insercional , Mutación , Fenotipo , Filogenia , Infertilidad Vegetal/genética , Raíces de Plantas/anatomía & histología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
20.
Plant Physiol ; 138(4): 2005-18, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16040664

RESUMEN

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiología , Polen/fisiología , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Mutagénesis Insercional , Mutación , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/química
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