Este trabalho foi financiado pelo programa de pesquisa do genoma de plantas da National Science Foundation (NSF-PGRP-IOS-1339388).

1. o crescimento das plantas
<ol><li>5-10 sementes de soja (82 Williams) em uma panela de 13 cm em estufa sob condições de longo-dia (16 h de luz em 1.500 µmol m-2 s-1) a 25 ° C, a mistura de solo personalizada da porca para soja (a 1: proporção de 1:1 de areia do solo, perlite e torpedo).</li>
</ol>2. preparação do ADN do plasmídeo
<ol><li>Usando a ponta da pipeta estéril ou palito de dente, escolher uma única colônia ou estoque de glicerol congelados de Escherichia coli , carregando o plasmídeo contendo o gene de interesse e inoculá-lo em 20 mL de meio líquido de Luria-Bertani (LB) com antibióticos apropriados em um balão de 50 mL.</li>
	<li>Incube o frasco a 37 ° C durante a noite em uma coqueteleira. Recolha as bactérias por centrifugação a suspensão a 12.000 x g, à temperatura ambiente por 5 min e descartar o sobrenadante. Extrair e purificar o plasmídeo seguindo o procedimento do fabricante de um kit de preparação do plasmídeo.</li>
</ol>3. isolamento de protoplastos
<ol><li>Corte recém-expandido unifoliate folhas de plântulas de soja 10 dias de idade (Figura 1). Nota: A seleção das folhas em uma fase do desenvolvimento é a chave para o sucesso de preparação de protoplastos de soja. Só use folhas apenas expandidas em estágios iniciais do desenvolvimento. Como folhas maduras, paredes celulares tornam-se mais difícil de digerir.</li>
	<li>Com uma lâmina de barbear fresca, retire a nervura central da folha unifoliate e em seguida, corte os restos em 0,5-1 mm tiras. Nota: Duas folhas unifoliate digeridas em 10 mL de solução de enzima dará protoplastas suficientes para mais de 10 transformações.</li>
	<li>Usando um par de pinças, transferência a folha retira imediatamente e suavemente em 10 mL de solução recentemente preparada de enzima (tabela 1) em um tubo de 15 mL. Vácuo infiltrar a folha tiras para 15 min à temperatura ambiente.</li>
	<li>Incube as tiras de folha na solução enzimática com agitação suave (40 rpm) sob condições de pouca luz para 4-6 h à temperatura ambiente. Certifique-se de que a solução de enzima ficar verde-amarelo, como os protoplastas são liberados. Verifique se a solução de enzima/protoplastas sob o microscópio (X10). Nota: Os protoplastas lançados são esféricos, enquanto as células não digeridas tem forma oval ou irregular.</li>
	<li>Transferir a 10 mL da solução de enzima/protoplastos em um tubo de 50ml derramando suavemente e adicionar 10 mL de solução de W5 (tabela 1) à temperatura ambiente para parar a digestão. Inverta o tubo suavemente várias vezes. Despeje delicadamente a solução enzimática/protoplastas em uma malha de nylon limpo 75 µm colocada em cima de um tubo de 50 mL para remover os tecidos da folha não digerido.</li>
	<li>Centrifugue a solução de fluxo através da enzima/protoplastas em 100 g de x no tubo de 50 mL por 1-2 min à temperatura ambiente. Remova cuidadosamente o sobrenadante usando uma pipeta sorológica 10ml sem perturbar o sedimento de protoplastos.</li>
	<li>Ressuspender os protoplastas e diluir para uma concentração de 2 x 105 mL-1 , na solução de W5 refrigerada a 4 ° C, contando número de protoplastos em um hemacytometer sob o microscópio (x10). Manter os protoplastas no gelo por 30 min.</li>
	<li>Centrifugar a suspensão de 100 g de x para 1-2 min à temperatura ambiente e remova cuidadosamente a solução W5 usando uma pipeta de 1ml sem perturbar o sedimento de protoplastos. Ressuspender os protoplastas em solução MMG (tabela 1) na concentração de 2 x 105 mL-1 , à temperatura ambiente.</li>
</ol>4. transformação de protoplastos
<ol><li>Faça 100 alíquotas µ l de protoplastas (2 x 104 protoplastas em 2 x 105 mL-1) em tubos de microcentrifuga de baixa adesão de 1,5 mL usando sem cortes pontas de pipetas 200 µ l. Colocar uma alíquota de lado para servir como controle negativo. Adicione 10 µ l de plasmídeo (10-20 µ g) em cada uma das parte alíquota do resto dos protoplastas.</li>
	<li>Lentamente, adicionar 110 µ l de solução recentemente preparada de PEG (tabela 1) na parede interna do tubo de microcentrifuga de 1,5 mL e depois inverta suavemente e rodar o tubo até que a solução se torne homogênea.</li>
	<li>Incube a mistura de transformação à temperatura ambiente por 15 min.</li>
	<li>Para parar a transformação, lentamente adicione 400 µ l de solução W5 no tubo de 1,5 mL à temperatura ambiente e inverta suavemente o tubo até que a solução se torne homogênea. Centrifugar o tubo a 100 g para 1-2 min à temperatura ambiente e descartar o sobrenadante com uma pipeta.</li>
	<li>Adicionar 1 mL de solução WI (tabela 1) para o tubo e ressuspender delicadamente pipetando 1 - 2 vezes. Adicione 1 mL de soro de 5% (vol/vol) bezerro estéril em cada poço de uma placa de cultura de tecidos de 6-poços para revestir a superfície e evitar os protoplastas de degola para a placa.</li>
	<li>Após alguns segundos, descarte o soro de vitela com uma pipeta. Transferi os protoplastas ressuspensão em um poço da placa de cultura. Cubra o prato com uma tampa.</li>
</ol>5. protoplastos incubação e colheita
<ol><li>Incube os protoplastas à temperatura ambiente durante 1-2 dias no escuro. Nota: Achamos que dois dias de incubação rende mais forte sinal de proteína fluorescente, em geral, em relação à incubação de um dia, e que o sinal de proteína fluorescente pode durar até 3-4 dias.</li>
	<li>Transferi a solução de protoplastos para um tubo de microcentrifugadora de baixa adesão de 1,5 mL. Centrifugar o tubo a 100 x g por 1-2 min à temperatura ambiente a colheita protoplastas. Remover o sobrenadante com uma pipeta e transferir 10 protoplastas µ l numa lâmina de vidro.</li>
	<li>Observe o sinal fluorescente sob fluorescência ou microscopia confocal. Use protoplastas não-transformadas como controlo negativo (nenhum sinal deve ser observado).</li>
</ol>

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Os autores declaram que eles têm não tem interesses financeiro concorrente.

Este protocolo para a isolação de protoplastas de soja e o aplicativo para estudos de expressão transiente tem sido exaustivamente testado e funciona muito bem em nosso laboratório. Os procedimentos são simples e fáceis e exigem equipamento normal e custo mínimo. Nosso protocolo produz grandes quantidades de protoplastas uniforme, de alta qualidade em comparação com métodos anteriormente relatados34,35,36,<s...

Protoplastas são células vegetais que possuem paredes celulares removidas. Como eles mantêm a maioria dos recursos e atividades das células vegetais, protoplastas são um bom modelo sistema para observar e avaliar diversos eventos celulares e são ferramentas valiosas para estudar a hibridação somática1 e regeneração2da planta. Protoplastas tem sido também amplamente utilizados para planta transformação3,4,5, desde paredes celulares caso contrário iria bloquear a passagem de DNA para a célula. Protoplastas de possuam algumas das respostas fisiológicas e processos celulares de plantas intactas, oferecendo, portanto, o valor fundamental na pesquisa básica para estudar a localização Subcellular da proteína6,7,8, de9,de interações proteína-proteína10e o promotor atividade11,12,13 em células vivem.
O isolamento dos protoplastas de planta foi primeiramente relatado em 196014 e os protocolos para isolamento e transformação de protoplastas foram desenvolvidos e optimizados. Um procedimento padrão de isolamento de protoplastos envolve o corte de folhas e digestão enzimática das paredes celulares, seguido de separação de protoplastas lançados de restos de tecido não-digeridos. Estratégias de transformação inclui electroporation15,16, microinjeção17,18e baseadas em polietileno glicol (PEG)4,5,19 métodos. Uma grande variedade de espécies têm sido relatados bem sucedida para o isolamento de protoplastos, incluindo cítricas20, Brassica21, Solanaceae22 e outras famílias de plantas ornamentais23,24. Enquanto o tecido diversos tipos são usados em várias espécies, um sistema de expressão transiente em protoplastos de mesofilo de Arabidopsis (TEAMP) isolado de folhas da planta modelo Arabidopsis thaliana tem sido bem estabelecida25 e amplamente adotado para diversas aplicações.
Feijão de soja (Glycine max (L.) Merr.) é uma das mais importantes proteínas e óleo para as culturas26. Ao contrário de Arabidopsis e arroz, obtenção de plantas transgênicas de soja é conhecido por ser um pouco difícil e baixa eficiência. Agrobacterium tumefaciens-mediada por infiltração foi usada popularmente para estudos de expressão transiente gene nas células epidérmicas em tabaco27 e mudas em Arabidopsis28,29, Considerando que Agrobacterium rhizogenes tem sido usado para transformação de raízes peludas na soja30. Abordagens de silenciamento de gene induzida por vírus têm sido utilizadas para downregulation de alvo genes31,32 e transiente expressão33 de forma sistêmica. Protoplastas fornecem uma alternativa valiosa e versátil para essas abordagens. Protoplastas podem ser obtidos de materiais na superfície do feijão de soja e permitam a expressão do transgene rápida e sincronizada. No entanto, desde o isolamento de sucesso inicial de protoplastas de soja no 198334, houve limitados relatórios sobre a aplicação da protoplastas em soja35,36,37, 38, principalmente devido à relativamente baixos rendimentos de protoplastas de soja.
Aqui, descrevemos um protocolo simples e eficiente para o isolamento de protoplastas de soja e sua aplicação para estudos de expressão transiente de gene. Usando o jovens unifoliate folhas de plântulas de soja, fomos capazes de obter grandes quantidades de protoplastas vitais dentro de algumas horas. Além disso, otimizamos a um método de transformação de PEG-cálcio-mediada que é simples e de baixo custo para entregar o DNA nas protoplastas de soja com alta eficiência.

<table><tbody><tr><td>MES</td><td>Sigma Aldrich</td><td>M8250-100G</td><td></td></tr><tr><td>Cellulase CELF</td><td>Worthington Biological Corporation</td><td>LS002611</td><td></td></tr><tr><td>Pectolyase Y-23</td><td>BioWorld</td><td>9033-35-6</td><td></td></tr><tr><td>CELLULASE &quot;ONOZUKA&quot; R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>MACEROZYME R-10</td><td>yakult</td><td>10g</td><td></td></tr><tr><td>Mannitol</td><td>ICN Biomedicals</td><td>152540</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C79-500g</td><td></td></tr><tr><td>BSA</td><td>NEB</td><td>R3535S</td><td></td></tr><tr><td>DTT</td><td>Sigma Aldrich</td><td>D5545-5G</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653-1kg</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217-500g</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266-100g</td><td></td></tr><tr><td>PEG4000</td><td>Fluka</td><td>81240</td><td></td></tr><tr><td>nylon mesh</td><td>carolina</td><td>652222N</td><td></td></tr><tr><td>Tissue Culture Plates</td><td>USA Scientific</td><td>CC7682-7506</td><td></td></tr><tr><td>Razor Blades</td><td>Fisher</td><td>12-640</td><td></td></tr><tr><td>hemacytometer</td><td>hausserscientific</td><td>1483</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EZNA plasmid miniprep kit</td><td>Omega</td><td>D6942-01</td><td></td></tr><tr><td>GeneJET Plasmid Miniprep Kit</td><td>Thermo Scientific</td><td>K0502</td><td></td></tr><tr><td>Centrifuge 5810</td><td>eppendorf</td><td>5811000827</td><td></td></tr><tr><td>Centrifuge 5424</td><td>eppendorf</td><td>22620401</td><td></td></tr><tr><td>Jencons Powerpette Plus Pipet Controller</td><td>Jencons</td><td>14526-202</td><td></td></tr><tr><td>Zeiss 710 Confocal Microscope</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td><td>Ambion</td><td>AM12450</td><td></td></tr><tr><td>15 mL Centrifuge Tubes</td><td>Denville</td><td>C1018-P</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville</td><td>C1060-P</td><td></td></tr><tr><td>Newborn Calf Serum</td><td>Thermo Scientific</td><td>16010159</td><td></td></tr><tr><td>Soil</td><td>Ingram&#039;s Nursery</td><td></td><td></td></tr><tr><td>perlite</td><td>Vigoro</td><td>100521091</td><td></td></tr><tr><td>Torpedo Sand</td><td>JKS Ventures</td><td></td><td></td></tr><tr><td>LB Broth, Lennox (Powder)</td><td>Fisher</td><td>BP1427-500</td><td></td></tr></tbody></table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

Feijão de soja (Glycine max (L.) Merr.) é uma espécie de cultura importante e tornou-se um modelo de vegetal para os estudos das vias genéticas e bioquímicas. Portanto, é importante estabelecer um sistema de expressão de gene transiente eficiente em soja. Aqui, nós relatamos um protocolo simples para a preparação de protoplastas de soja e sua aplicação para análises funcionais transitórias. Nós achamos que o jovens unifoliate folhas de plântulas de soja resultaram em grandes quantidades de protoplastas de alta qualidade. Otimizando um método de transformação PEG-cálcio-mediada, atingimos a eficiência elevada de transformação usando protoplastas de unifoliate de soja. Este sistema fornece um modelo eficiente e versátil para exame dos complexos mecanismos de regulamentação e sinalização em células vivas de soja e pode ajudar a melhor compreender diversos processos celulares, fisiológicos e de desenvolvimento das leguminosas.

Diferentes órgãos de soja 10 dia de idade foram testados para a preparação de protoplastos (Figura 1) e rendimentos foram observados ao microscópio (Figura 2). Paredes celulares do hypocotyl e epicotyl foram mal digeridas, e algumas células ficaram anexadas ao outro (Figura 2B, 2C). No cotilédone (Figura 2D) e raiz (Figura 2A<...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Desenvolvemos um protocolo simples e eficiente para a preparação de grandes quantidades de protoplastas de soja estudar mecanismos complexos regulatórios e sinalização em células vivas.

Um método simples para o isolamento de aplicativo para análises de expressão de Gene transiente e protoplastas de soja

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Research

JoVE Journal

Biology

24.6K visualizações.  California State University, Northridge. Desenvolvemos um protocolo simples e eficiente para a preparação de grandes quantidades de protoplastas de soja estudar mecanismos complexos regulatórios e sinalização em células vivas.

Vídeo: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

A plataforma robótica de alta capacidade de protoplastos Isolamento e Transformação

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genética

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Expressão Gênica transiente em tabaco usando Assembleia Gibson eo Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Ambiente

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

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica e Análise de Imagem Dinâmica de Estudos de Expressão Gênica em Tecidos Vegetais

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Identificação de sequências de localização Plasmodesmal em proteínas em Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Imunologia e Infecção

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 ...

Produção de alta eficiência de raízes pilosas de soja transgênica

Soybean Hairy Root Transformation for the Analysis of Gene Function

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with soil-borne microbes that form symbiosis to fix nitrogen and pathogens, which makes research involving soybean root genetics of prime importance to improve its agricultural production. The genetic transformation of soybean hairy roots (HRs) is mediated by the Agrobacterium rhizogenes strain NCPPB2659 (K599) and is an efficient tool for studying gene function in soybean roots, taking only 2 months from start to finish. Here, we provide a detailed protocol that outlines the method for overexpressing and silencing a gene of interest in soybean HRs. This methodology includes soybean seed sterilization, infection of cotyledons with K599, and the selection and harvesting of genetically transformed HRs for RNA isolation and, if warranted, metabolite analyses. The throughput of the approach is sufficient to simultaneously study several genes or networks and could determine the optimal engineering strategies prior to committing to long-term stable transformation approaches.

Autor em destaque: Transformação da raiz peluda da soja para a análise da função gênica  

Here, we present a protocol for the high-efficiency production of transgenic soybean hairy roots.

Transformação de raízes pilosas de soja para análise da função gênica

Soybean (Glycine max) is a valuable crop in agriculture that has thousands of industrial uses. Soybean roots are the primary site of interaction with ...

Um método simples para o isolamento de aplicativo para análises de expressão de Gene transiente e protoplastas de soja

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