Name: quantification of endogenous auxin and cytokinin during internode culture of ipecac
Uploaded: 2018-03-15T14:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Nós estamos gratos ao Sr. Akira Murakami do departamento de Biociências aplicadas, Universidade de Toyo e Sr. Koudai Taniguchi do centro de tecnologia agrícola de Gunma para sua assistência técnica. Nós também estamos gratos ao Professor Shosaku Kashiwada e Dr. Uma Maheswari Rajagopalan, Universidade Toyo para suas sugestões. Este estudo foi suportado em parte pelo centro de pesquisa para a vida e ciências ambientais, Universidade de Toyo.

Nota: Poaia (ipecacuanha c.) foi usada neste estudo porque facilita a análise de fitohormônios endógenas.
1. crescimento condições para induzir brotos adventícios de ipeca
<ol><li>Prepare phytohormone livre B5 médio ajustado ao pH 5.710e adicionar gelano 0,2%. Esterilize em autoclave.</li>
	<li>Despeje uma placa de Petri estéril (90 mm × 20 mm), 25 mL do meio de autoclave.</li>
	<li>Corte-8mm internodal segmentos de plântulas de ipecac, usando um bisturi cirúrgico com uma lâmina n º 22, em uma placa de acrílico estéril e coloque no meio (Figura 1).</li>
	<li>Cultura livre phytohormone B5 meio a 24 ° C sob 15 µmol m− 2 s− 1 de luz em um ciclo escuro da luz/10-h 14-h por 5 semanas.</li>
	<li>Identificar regiões eu (apical) a IV (basal) em cada segmento (Figura 1B). Conte o número de brotos adventícios > 0,3 mm de comprimento em cada região sob um microscópio, uma vez por semana.</li>
</ol>2. extração e purificação de fitohormônios
<ol><li>Coloque uma pérola zircônia 5 mm em cada um dos quatro tubos 2 mL de amostra.</li>
	<li>Corte os segmentos em regiões I a IV (cada 2 mm em comprimento) usando um bisturi cirúrgico em uma placa de acrílico estéril.</li>
	<li>Colete oito segmentos de cada região em tubos de amostra separada (peso fresco de 10 a 30 mg).</li>
	<li>Pesar e em seguida, congelar as amostras em nitrogênio líquido.</li>
	<li>Esmaga as amostras congeladas, usando um homogeneizador de grânulo-baseado.</li>
	<li>Suspender as amostras esmagadas em 1 mL acetonitrila contendo 500 pg de cada padrão interno de auxina e CKs (d5-IAA, d5- tZ, d5- tZR, d6- iP, d6- iPR) usando um misturador do vortex.</li>
	<li>Mantenha a 4 ° C, durante 1 h e, em seguida, centrifugar a 3.500 × g por 5 min à temperatura ambiente.</li>
	<li>Lave o pellet em acetonitrila 80% (v/v) contendo ácido acético a 1% (v/v). Centrifugar novamente 3.500 × g por 5 min à temperatura ambiente. Combine os sobrenadantes (a partir de etapas 2.7 e 2.8) em um tubo de vidro descartáveis.</li>
	<li>Adicione 600 µ l contendo ácido acético de 1% (v/v) para cada sobrenadante combinada de água e evaporar o acetonitrilo usando um concentrador a vácuo.</li>
	<li>Equilibrar o hidrófilo lipofílico-balanceamento de cartuchos de coluna (HLB) aplicando-se 1 mL de água contendo 1% (v/v) de ácido acético, metanol e acetonitrila.</li>
	<li>Aplica uma solução de amostra por cartucho HLB equilibrado.</li>
	<li>Lave os cartuchos com 1 mL de água contendo ácido acético a 1% (v/v).</li>
	<li>Eluir todos hormônios com acetonitrilo de 80% (v/v) de 2 mL contendo ácido acético a 1% (v/v) em um tubo de vidro.</li>
	<li>Evapore o acetonitrilo no eluato para obter o extrato em água contendo 1% (v/v) de ácido acético usando um concentrador a vácuo. Nota: Não seque isto completamente.</li>
	<li>Equilibrar os cartuchos de coluna de modo misto, forte permutadora de catiões (MCX) aplicando-se 1 mL acetonitrilo, 1 mL de metanol, 0,5 mL 0,1 M de HCl e 1 mL de água contendo ácido acético a 1% (v/v).</li>
	<li>Aplica uma solução de amostra por cartucho MCX equilibrado.</li>
	<li>Lave os cartuchos com 1 mL de água contendo ácido acético a 1% (v/v).</li>
	<li>Eluir IAA com acetonitrilo de 30% (v/v) de 2 mL contendo ácido acético a 1% (v/v) em um tubo de vidro.</li>
	<li>Lave os cartuchos com acetonitrilo de 80% (v/v) de 2 mL contendo 1% (v/v) de ácido acético.</li>
	<li>Lave os cartuchos com 2 mL de água e 1 mL de água contendo amônia aquosa de 5%.</li>
	<li>Eluir os CKs com acetonitrilo de 60% (v/v) de 2 mL contendo 5% de amônia aquosa em um tubo de vidro.</li>
	<li>Evaporar o solvente de cada fração do hormônio usando um concentrador a vácuo e armazenar a 30 ° C até a análise de LC-MS/MS.</li>
</ol>3. a LC-MS/MS análise de IAA e CKs
<ol><li>Dissolver cada extracto de hormônio em um tubo com 600 µ l de metanol e transferir a solução para um frasco de recuperação total do pescoço de parafuso. Evapore o solvente usando um concentrador a vácuo.</li>
	<li>Dissolva a fração de IAA em 20 µ l de 30% (v/v) acetonitrila e as frações CK em água de 20 µ l contendo ácido acético a 1% (v/v) em frascos de recuperação total de pescoço de parafuso.</li>
	<li>Analise as amostras no modo de íon positivo em um sistema de MS triplo quadrupolo equipado com um sistema HPLC. Nota: Podemos definir as condições HPLC conforme listado na tabela 1.<ol>
<li>Para a eluição do IAA, use um gradiente binário de 5% - 50% solvente B mais 7 min, em seguida, aumentar por 98% de solvente B Aguarde 1 min e então equilibrar por 2 min em 5% solvente B por injeção de ninho.</li>
		<li>Para a eluição de CK, use um gradiente binário de 2% - 40% de solvente B mais 5 min, 40% - 70% de solvente B em 7 min, em seguida, aumentar em 95% solvente B Aguarde 1 min e então equilibrar por 2 min em 2% solvente B por ninho de injeção.</li>
		<li>Definir a ionização electrospray (ESI)-MS parâmetros da fonte de íons, conforme listado na tabela 2.</li>
	</ol>
</li>
	<li>Use a reação múltiplo monitoramento transição (MRM) para quantificação de cada analito listado na tabela 3.</li>
	<li>Quantificar os níveis endógenos de IAA e CK contra uma curva-padrão do rácio de sem rótulo para padrões de deutério-rotulado.</li>
</ol>

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Os autores declaram que têm não há conflitos de interesse.

Para identificar a distribuição dos fitohormônios envolvidos na organogênese, é importante o uso de materiais vegetais em que organogênese pode ser observado na média phytohormone-livre, porque quando os fitohormônios são exogenamente aplicados a explantes para induzir brotos ou raízes, elas afetam o explante todo, tornando-se difícil avaliar a plasticidade inerente de plantas na diferenciação celular e organogênese. Brotos adventícios podem ser induzidos em meios de cultura livre phytohormone em outras es...

Gottlieb Haberlandt (1854-1945) propôs o conceito de "totipotência", pela qual planta células podem dividir, diferenciar e regenerar plantas inteiras mesmo após sua prévia diferenciação em tipos de células específicas em plantas maduras1. Em cultura de tecidos, se planta regeneração pode ser induzida ou não é determinada pela combinação e concentração de fitohormônios exogenamente aplicadas no meio de crescimento. Skoog e Miller encontraram que brotos adventícios poderiam ser induzidos de tabaco calo no meio de cultura contendo uma alta proporção de CKs de auxinas, Considerando que raízes adventícias poderiam ser induzidas em meio contendo uma baixa relação2. Desde essa constatação, cultura de tecidos foi amplamente utilizada para a propagação de culturas economicamente importantes e a regeneração de plantas transgénicas3. Brotos adventícios podem ser induzidos a partir de tecidos que não sejam o meristema apical de tiro, como folhas, raízes e entrenós. Tratamento de phytohormone é necessário para a indução de brotos adventícios na maioria das espécies de plantas. No entanto, o ideais concentrações e combinações diferem por espécie e entre tecidos utilizados como explantes. Assim, muito esforço vai para determinação da concentração ideal e combinações de fitohormônios para experimentos.
Carapichea ipecacuanha (Brot.) L. Andersson (ipecacuanha) é uma planta medicinal que contém alcaloides como Emetina e a Cefalina, principalmente no raízes4. Extratos de raiz são usados como um expectorante, um emético e um amoebicide5. Embora ipecac cresce naturalmente nas florestas tropicais do Brasil, é relutante em conjunto de sementes na cultura, e a taxa de germinação diminui durante o armazenamento de sementes no Japão, com seus de clima mais frio6. Em vez disso, ele é propagado por cultura de tecidos, no qual adventícios atirar formação em entrenós é o mais eficiente método de7,8. Curiosamente, os brotos adventícios podem ser induzidos nesta espécie sem phytohormone tratamento8.
Brotos adventícios são formados na epiderme na região apical dos segmentos internodal sem callusing, mas não na região basal9. Esta diferença indica a polaridade do tecido em segmentos internodal, que é, provavelmente, ao abrigo do Regulamento fitohormonal. O sistema de cultura de ipecac permite uma oportunidade única para analisar as alterações nos níveis endógenos phytohormone durante formação de tiro acidental. Aqui apresentamos nosso método para a análise dos níveis endógenos de uma auxina (ácido indol-3-acético (IAA)) e quatro CKs (isopentenilo adenina (iP), Ribosídica de adenina de isopentenilo (iPR), trans-Zeatina (tZ) e trans-Zeatina Ribosídica (tZR)) em segmentos internodal através do uso de LC-MS/MS.

<table><tbody><tr><td>[2H5]indole-3-acetic acid</td><td>Olchemlm Ltd</td><td>031 1531</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin</td><td>Olchemlm Ltd</td><td>030 0301</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin riboside</td><td>Olchemlm Ltd</td><td>030 0311</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenine</td><td>Olchemlm Ltd</td><td>030 0161</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenosine</td><td>Olchemlm Ltd</td><td>030 0171</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>indole-3-acetic acid</td><td>Wako</td><td>098 00181</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin</td><td>SIGMA-ALDRICH</td><td>Z0876 5MG</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin riboside</td><td>Wako</td><td>262 01081</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenine</td><td>SIGMA-ALDRICH</td><td>D7674 1G</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenosine</td><td>ACROS ORGANICS</td><td>22648 1000</td><td>standard for LC-MS/MS</td></tr><tr><td>acetonitrile hypergrade for LC-MS LiChrosolv</td><td>MERCK</td><td>1.00029.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>Water for chromatography LiChrosolv</td><td>MERCK</td><td>1.15333.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>HPLC</td><td>SHIMADZU</td><td>Prominence</td><td></td></tr><tr><td>MS</td><td>Sciex</td><td>3200QTRAP</td><td></td></tr><tr><td>Oasis HLB 30 mg/1 cc</td><td>Waters</td><td>WAT094225</td><td>cartridge column</td></tr><tr><td>Oasis MCX 30 mg/1 cc</td><td>Waters</td><td>186000252</td><td>cartridge column</td></tr><tr><td>screw neck total recovery vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td><td>Waters</td><td>186000274</td><td></td></tr><tr><td>Acquity UPLC BEH C18, 2.1x100 mm</td><td>Waters</td><td>186002350</td><td>UPLC column</td></tr><tr><td>Proshell 120 EC-C18, 2.1x50 mm</td><td>Agilent</td><td>699775-902</td><td>UPLC column</td></tr><tr><td>Digital microscope</td><td>Leica</td><td>DHS1000</td><td></td></tr><tr><td>TissueLyser II</td><td>QIAGEN</td><td>85300</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No. 22</td><td></td></tr><tr><td>Scalpel handle</td><td>Feather</td><td>No. 4</td><td></td></tr><tr><td>Savant SpeedVac/Refregerated vapor trap</td><td>Thermo Fisher Scientific</td><td>SPD111/RVT4104</td><td>vacuum concentrartor</td></tr><tr><td>Disposable glass tobe (13x100 mm)</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>Sterile petri dish</td><td>INA OPTICA</td><td>I-90-20</td><td></td></tr></tbody></table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

Formação de tiro acidental é uma técnica importante para a propagação de culturas economicamente importantes e a regeneração de plantas transgênicas. Tratamento de phytohormone é necessário para a indução de brotos adventícios na maioria das espécies. Se os brotos adventícios podem ser induzidos é determinado pelo saldo entre a auxina e citocinina (CK) níveis. Muito esforço vai para determinar concentrações óptimas e combinações de fitohormônios em cada tecido usado como explantes e em cada espécie de planta. Em ipecac, no entanto, brotos adventícios podem ser induzidos em segmentos internodal em meio de cultura sem tratamento phytohormone. Isso permite que a plasticidade inerente de ipeca para diferenciação de célula a ser avaliada. Para induzir os brotos adventícios em ipecac, nós cultivadas internodal segmentos a 24 ° C sob 15 µmol m− 2 s− 1 de luz em um ciclo escuro da luz/10-h 14-h phytohormone livre B5 meio solidificado com 0,2% gelano por 5 semanas. Para investigar a dinâmica phytohormone durante formação de tiro acidental, medimos endógeno ácido indol-3-acético e CKs nos segmentos por líquido espectrometria de massa em tandem-cromatografia de LC-MS/MS. Este método permite a análise de ácido indol-3-acético endógeno e CKs níveis de forma simples. Pode ser aplicado para investigar a dinâmica de auxina endógena e CK durante a organogênese em outras espécies de plantas.

Em 1 semanast , sem brotos adventícios tinham formado. Na semana de 2nd , pequenos brotos apareceram. O 3rd e 4 semanas deth , o número de tiros aumento principalmente nas regiões apicais (I e II)(Figura 2). Na semana de 5th , o número de brotos foi aproximadamente 7 em região eu e 5 na região II (Figura 2B). Em contraste, apenas alguns...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Quantificação de auxina endógena e citocinina durante Internode cultura de ipeca

Brotos adventícios podem ser induzidos em segmentos internodal de ipeca sem tratamento phytohormone. Para avaliar a dinâmica phytohormone durante formação de tiro acidental, medimos endógena auxina e citocinina em segmentos internodal por LC-MS/MS.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

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9.0K visualizações.  Toyo University. Brotos adventícios podem ser induzidos em segmentos internodal de ipeca sem tratamento phytohormone. Para avaliar a dinâmica phytohormone durante formação de tiro acidental, medimos endógena auxina e citocinina em segmentos internodal por LC-MS/MS.

Vídeo: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Utilizando o composto libertador de etileno, ácido 2-cloroetilfosfônico, como uma ferramenta para estudar a resposta do etileno em bactérias

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Bioquímica

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

Genética

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

Extração e Quantificação de Polifosfatos inositol solúveis e radiolabelados de diferentes espécies vegetais usando SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

Ajustando a degradação para conseguir a depleção de proteína específica e eficiente

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

Imunologia e Infecção

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

Sistema induzível de raiz lateral em Arabidopsis e milho

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

Biologia

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

Uma estratégia para validar o papel da mediada por calejado Plasmodesmal Gating na Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

Um sistema de co-cultivo hidropônico para análise simultânea e sistemática de interações e sinalização moleculares de plantas/microorganismos

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplicação por citometria de fluxo de protoplastas de tubérculos isolados de medição

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Identificação dos genes envolvidos no desenvolvimento estomático via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Imagem de alta resolução da biossíntese de clorofila em mudas de Arabidopsis

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Autor em destaque: medição não invasiva de alta resolução da síntese de clorofila durante o desestiolamento

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Ensaio Não Invasivo para Determinação da Cinética de Biossíntese de Clorofila durante os Estágios Iniciais da Desetiolação de Arabidopsis

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

Quantificação de auxina endógena e citocinina durante Internode cultura de ipeca

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Utilizando o composto libertador de etileno, ácido 2-cloroetilfosfônico, como uma ferramenta para estudar a resposta do etileno em bactérias

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Extração e Quantificação de Polifosfatos inositol solúveis e radiolabelados de diferentes espécies vegetais usando SAX-HPLC

Ajustando a degradação para conseguir a depleção de proteína específica e eficiente

Sistema induzível de raiz lateral em Arabidopsis e milho

Uma estratégia para validar o papel da mediada por calejado Plasmodesmal Gating na Tropic Response

Um sistema de co-cultivo hidropônico para análise simultânea e sistemática de interações e sinalização moleculares de plantas/microorganismos

Endoreduplicação por citometria de fluxo de protoplastas de tubérculos isolados de medição

Identificação dos genes envolvidos no desenvolvimento estomático via Epidermal Phenotype Scoring

Ensaio Não Invasivo para Determinação da Cinética de Biossíntese de Clorofila durante os Estágios Iniciais da Desetiolação de Arabidopsis