Somos gratos a Sabin Russell para a assistência edição. Este trabalho fez parte do Instituto Joint BioEnergy DOE (http://www.jbei.org), apoiado pelo Departamento de Energia dos EUA, Escritório de Ciência, Instituto de Pesquisa Ambiental e Biológica, por meio do contrato DE-AC02-05CH11231 entre Lawrence Berkeley Laboratório Nacional e do Departamento de Energia dos EUA.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+in+the+interaction+of+direct+dyes+with+polysaccharides.">Specificity in the interaction of direct dyes with polysaccharides.</a> Carbohydrate Research. 85, 271-287 (1980).</li><li>Hughes, J., McCully, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+an+optical+brightener+in+the+study+of+plant+structure.">The use of an optical brightener in the study of plant structure.</a> Stain technology. 50, 319-329 (1975).</li><li>Verbelen, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapsed+xylem+phenotype+of+Arabidopsis+identifies+mutants+deficient+in+cellulose+deposition+in+the+secondary+cell+wall).">Collapsed xylem phenotype of Arabidopsis identifies mutants deficient in cellulose deposition in the secondary cell wall).</a> The Plant Cell Online. 9, 689-701 (1997).</li><li>Iiyama, K., Pant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+M&#228;ule+colour+reaction.+Introduction+of+methylated+syringyl+nuclei+into+softwood+lignin.">The mechanism of the M&#228;ule colour reaction. Introduction of methylated syringyl nuclei into softwood lignin.</a> Wood Sci. Technol. 22, 167-175 (1988).</li><li>Yang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+secondary+cell+wall+deposition+in+plants.">Engineering secondary cell wall deposition in plants.</a> Plant biotechnology journal. 11, 325-335 (2013).</li><li>Zhong, R., Ripperger, A., Ye, Z. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+deposition+of+lignin+in+the+pith+of+stems+of+two+Arabidopsis+mutants.">Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants.</a> Plant physiology. 123, 59-70 (2000).</li><li>Chapple, C. C., Vogt, T., Ellis, B. E., Somerville, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Arabidopsis+mutant+defective+in+the+general+phenylpropanoid+pathway.">An Arabidopsis mutant defective in the general phenylpropanoid pathway.</a> The Plant cell. 4, 1413-1424 (1992).</li><li>Fagard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PROCUSTE1+Encodes+a+Cellulose+Synthase+Required+for+Normal+Cell+Elongation+Specifically+in+Roots+and+Dark-Grown+Hypocotyls+of+Arabidopsis.">PROCUSTE1 Encodes a Cellulose Synthase Required for Normal Cell Elongation Specifically in Roots and Dark-Grown Hypocotyls of Arabidopsis.</a> The Plant Cell Online. 12, 2409-2423 (2000).</li></ol>

The authors have no financial conflicts to disclose.

Secções tronco de A. thaliana são amplamente usados ​​para o estudo da organização das células da parede celular secundária e para examinar qualitativamente as diferenças entre o tipo selvagem e plantas transgénicas. As técnicas comumente utilizadas para a secção dos espécimes são corte manual direta; ou quando as amostras são incorporados em agarose ou fixador, o seccionamento pode ser feito com um vibratome ou micrótomo. Em contraste com o lado de corte, os dois últimos permitem reduzir o...

A parede celular da planta tem uma infinidade de informações dentro de seus vários componentes: lignina, celulose, hemicelulose (xilana, glucuronoxylan, xiloglucano, arabinoxilano, ligação mista glucana, ou glucomanana) e pectina. Técnicas histológicas fornecer dicas visuais importantes no estudo das diferenças dentro das paredes celulares secundárias a nível da organização e celulares. Várias técnicas histológicas foram desenvolvidos e podem ser encontrados na literatura, mas estas técnicas pode ser um desafio para principiantes e demorada porque os protocolos muito detalhado com instruções visuais simples são raramente, se alguma vez disponível. O objetivo deste estudo é fornecer orientações simples para técnicas de coloração histológicas para obter imagens de alta qualidade. Seccionamento do tecido do tronco é o primeiro passo para a visualização de células e paredes celulares de formas. Embora seções cortados à mão são baratos e levam menos tempo para se preparar, o uso de um vibratome oferece consistência eproduz imagens de alta qualidade. Usando um vibratome permite produzir melhor qualidade de dados, gerando até secções com a mesma espessura, o que ajuda a produzir imagens nítidas e reduz significativamente o risco de produzir diferenças entre amostras imprecisos que seria simplesmente causada por uma preparação de amostra mau. Usando resinas para fixar espécimes frescos pode ser um desafio para iniciantes e ainda pode ser demorado, mesmo para especialistas, quando uma análise precisa ser feito rapidamente. Além disso, torna-se impossível de medir qualquer actividade biológica com a amostra quando tiver sido incorporado numa resina. Uma técnica simples que emprega um molde de agarose e caseiro é útil para a incorporação de tecidos do caule, e que também pode ser utilizado em outras aplicações que requerem uma dissecção de tecidos moles. Em comparação com a incorporação de espécimes em resina, este método tem a vantagem de manter os tecidos vivos e reduzindo a manipulação da amostra. Seccionamento dos tecidos por meio de um vibratome é altamente preciso e gera homogeneaous secções, que, dependendo do objectivo do estudo, poderá, então, ser usados ​​com várias técnicas de coloração diferentes. Os métodos mais simples de visualizar lignina e outros compostos aromáticos empregar ultravioleta (UV). Excitação de moléculas aromáticas à base de luz UV é uma técnica antiga, mas ainda é uma das abordagens mais rápido para visualização de lignina. No entanto, a visualização UV na verdade não é ideal para a detecção de lignina porque a luz UV vão animar outros compostos aromáticos. A lignina é composta basicamente de três blocos de construção, as monolignóis (álcoois hidroxicinamil: álcool coniferil, álcool sinapyl e álcool p-cumaril) 1-3. Floroglucinol mancha pode proporcionar pistas para a extensão da cinnamaldehydes presentes no xilema, fibras e tecidos da traqueia 4. Floroglucinol é um bom indicador de cinnamaldehydes gerais e pode diferenciar entre cinnamaldehydes e outros aromáticos. Os monómeros de álcool sinapyl pode ser detectadae diferenciando-se pelo uso de Maule mancha. Azul de toluidina O é um corante policromático e, portanto, tem a capacidade de manchar os diferentes elementos da parede celular em diferentes cores 5,6. O principal uso do azul de toluidina O é detectar pectina e lignina 5,6. A vantagem do uso de azul de toluidina O é que muitos elementos da parede da célula pode ser visualizado num único passo. Ambos calcoflúor branco e vermelho congo são fáceis de trabalhar e pode ser usado para visualizar celulose. Calcofluor manchas brancas celulose, calose, e outros β-glucanas não-substituídos ou substituídos fracamente 6-9, ao passo que congo manchas vermelhas directamente para β-(1 → 4)-glucanos e em particular a celulose 10,11. O objetivo deste estudo é fornecer orientações simples para o uso das técnicas de coloração acima mencionados para obter imagens de alta qualidade a partir de A. thaliana hastes.

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			<td>EMD</td>
			<td>MERC2125</td>
			<td>CAS Number: 9012-36-6</td>
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			<td>Phloroglucinol</td>
			<td>Sigma</td>
			<td>P 3502</td>
			<td>1,3,5-trihydroxybenzene [CAS Number: 108-73-6]</td>
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			<td>Hydrochloric Acid</td>
			<td>EMD</td>
			<td>HX0603-75</td>
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			<td>EMD</td>
			<td>AX1303-6</td>
			<td>CAS Number: 1336-21-6</td>
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			<td>Sigma</td>
			<td>T3260</td>
			<td>Blutene chloride, Tolonium Chloride [CAS Number 92-31-9]&#160;</td>
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			<td>223468</td>
			<td>CAS Number 7722-64-7&#160;</td>
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			<td>KOPTEC</td>
			<td>V1401</td>
			<td>CAS Number: 64-17-5</td>
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		<tr>
			<td>Congo Red</td>
			<td>Sigma&#160;</td>
			<td>C6277</td>
			<td>Disodium 3,3&#39;-[[1,1&#39;-biphenyl]-4,4&#39;-diylbis(azo)]bis(4-aminonaphthalene 1-sulphonate) [CAS Number 573-58-0 ]</td>
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			<td>Stainless Steel Double Edge Blade (Personna Super)</td>
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			<td>NN-SD762S</td>
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			<td>Camera with CCD chip with no mechanical shutter&#160;</td>
			<td>Hamamatsu</td>
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			<td>QImaging</td>
			<td>MicroPublisher 5.0 RTV</td>
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			<td>Camera software&#160;</td>
			<td>&#160;Molecular Devices</td>
			<td>MetaMorph version 7.7.0.0</td>
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			<td>Imagining anaylsis&#160;</td>
			<td>Adobe&#160;</td>
			<td>Photoshop CS4</td>
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			<td>VWR</td>
			<td>48366-067</td>
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histochemical staining of arabidopsis thaliana secondary cell wall elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Stem Embedding e Seccionamento: A utilização do molde de plástico caseiro para incorporar os caules em 7% de agarose revelou-se fácil e rapidamente (Figura 1). As duas partes (A e B; Figura 1) do sistema de frasco incorporado torná-lo simples para liberar facilmente a haste incorporado em agarose como a agarose não se ater às partes dos frascos que são inertes, mantendo o sistema limpo. Os frascos podem ser reutilizados várias vezes durante anos. A conveniência de armaz...

Watch this Scientific Journal Video about Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements at JoVE.com

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Coloração histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celular Secundária parede Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

histochemical-staining-arabidopsis-thaliana-secondary-cell-wall

Research

JoVE Journal

Biology

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

40.1K visualizações.  Joint Bioenergy Institute. Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Vídeo: Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Isolamento de Protoplastos de tecidos de 14 dias de idade, mudas de Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Biologia

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-dip Transformação de Arabidopsis thaliana Examine a PTSO2:: β-glucuronidase Expressão Gênica Reporter

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Isolamento eficiente e rápida de embriões em início de carreira a partir de<em&gt; Arabidopsis thaliana</em&gt; Sementes

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

Análise do comportamento de crescimento de Arabidopsis thaliana em diferentes qualidades de luz

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

Investigação espaço-temporal da dinâmica ERL1 via imagem confocal

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Autor em destaque: Métodos baseados em imagens para estudar eventos de tráfego de membrana em células de linhagem estomática  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Métodos Baseados em Imagem para Estudo de Eventos de Tráfego de Membranas em Células de Linhagem Estomática

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Um método aprimorado para medir os perfis de hidratação do pólen em Arabidopsis thaliana

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Autor em destaque: Um bioensaio de hidratação de pólen in vivo de alta resolução, grão único para Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Bioensaio de Hidratação Polínica In Vivo de Grão Único de Alta Resolução para Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...