Gli autori desiderano ringraziare il Prof. Nicola Smirnoff dell&#39;Università di Exeter per i suoi consigli e l&#39;Università di meccanismo di Bioimmagini Exeter. Il finanziamento è stato fornito dal Regno Unito Biotecnologie e Scienze Biologiche Research Council (sovvenzione di riferimento BB/E002682/1).

Il protocollo indicati di seguito descrive un metodo semplice per l&#39;utilizzo di PFD come mezzo infiltrativa di montaggio in foglie di Arabidopsis thaliana, ma possiamo anticipare che questo metodo può essere utilizzato in una varietà di applicazioni di imaging in cui si desidera l&#39;aria ricca di tessuti. 1. Campioni di foglie di montaggio in PFD <ol><li> Preparare un vetrino da microscopio con un gas-permeabili guarnizione di polidimetilsilossano (PDMS, Carolina Gel di osservazione). PDMS è un polimero viscoelastico e può essere modellata per fornire una camera di misura per esigenze sperimentali. </li><li> Equilibrare PFD con l&#39;aria. Ciò può essere ottenuto facendo gorgogliare aria o agitando un piccolo volume di PFD in un aria piena di bottiglia. </li><li> Decantare PFD in un piatto aperto Petri e galleggiare una piantina intera o foglia asportato il PFD per 5 minuti. Il tessuto deve diventare trasparente, che ricorda di tessuto vetrificato (Figura 2 (a)). Le foglie possono apparire più scura o più chiara rispetto a prima esposizione a PFD, dipendenzading in condizioni di illuminazione e l&#39;età dei tessuti utilizzati. </li><li> Riempire la camera di PDMS di aria equilibrato PFD e con attenzione trasferire i campioni di tessuto dal piatto di incubazione Petri alla camera di PDMS. Sigillare il vetrino con un coprioggetto e l&#39;immagine a seconda delle esigenze sperimentali. </li><li> Nota: PFD funzioni anche bene in una camera aperta costruita su un vetrino o in una camera di perfusione ed è compatibile con grasso al silicone. PFD non è compatibile con i componenti in Teflon, come li dissolve. </li></ol> 2. Rappresentante dei risultati: Ispezione al microscopio di PFD-incubate campioni di foglie dimostra che la maggior parte di spazi aerei sono inondate. Figura 2 (b) mostra spazi aerei inondato di ricombinanti GFP sospeso in PFD. E &#39;evidente che la foglia è invaso su incubazione con PFD, ma che occasionalmente sacche d&#39;aria può rimanere. L&#39;acqua non inondare la foglia in queste condizioni. LSCM dei campioni incubati e montato in una<html> ir, acqua o spettacoli PFD PFD che dà un vantaggio per l&#39;acqua e l&#39;aria nella profondità delle immagini possibili (Figura 3). L&#39;esempio riportato in Figura 3 mostra Arabidopsis aria, acqua e PFD montato lascia esprimere cytosolically localizzato Venere, una variante di YFP 11 che si esprime costitutivamente sotto il promotore 35S. Possiamo vedere di circa 2 volte maggiore profondità delle immagini quando si confrontano PFD e acqua. Va notato, tuttavia, che il miglioramento preciso visto quando si usa PFD varia con apertura numerica della lente e il tipo di tessuto utilizzato. Abbiamo visto in streaming citoplasmatici e capelli allungamento radice in PFD piantine trattate, ognuno indicativo di piante sane. Inoltre abbiamo dimostrato che 2 Fv / Fm, una misura del funzionamento di fotosintesi delle piante 12 rimane entro limiti tollerabili su scale temporali utilizzati nella diagnostica per immagini. 3394fig1.jpg &quot;/&gt; Figura 1. Impianto di complessità e le implicazioni foglia ottica Diagrammatical rappresentazione che mostra le caratteristiche anatomiche della A. foglia thaliana in relazione al set-up ottico. Abbreviazioni usate sono obj. = Lente dell&#39;obiettivo, imm. = Fluido immersione, cov. = Coprioggetti, mnt. = Montaggio, taglio. = Cuticola, annuncio. ep. = Epidermide adaxial, st. = Poro stomatica, sp. = Mesofillo spugnoso, come = spazio aereo, amico. = Palizzata mesofillo, vb = fascio vascolare, annuncio. ep. = Epidermide adaxial. Pareti cellulari sono indicati da linee nere. <img alt="Figura 2." src="/files/ftp_upload/3394/3394fig2.jpg" /> Figura 2. PFD penetra facilmente foglie di Arabidopsis (A) Foglie incubate in aria, acqua o PFD per 5 minuti e ripreso con una fotocamera digitale Leica DCF3000FX con un microscopio Leica MZ16F (Leica Microsystems (UK) Ltd., Milton-Keynes, UK). Il logo Giove era stampato su pellicola di acetato ed illuminato indietrom al di sotto con una scatola di luce. Tempo di esposizione è stato di 89 ms e tutte le immagini sono stati raccolti e trattati in modo identico. Barre di scala rappresentano 2 mm. (B) PFD portando ricombinante purificata proteina verde fluorescente (GFP), delimita spazi aerei in vivo. GFP è falso colore verde e rosso è usato per mostrare l&#39;auto-fluorescenza della clorofilla, che delimita le cellule del mesofillo. (Figura 2 (b) riprodotto con il permesso e tutti i dettagli tecnici disponibili in Littlejohn et al. 2010 2). Barre di scala rappresentano il 25 m. <img alt="Figura 3." src="/files/ftp_upload/3394/3394fig3.jpg" /> Figura 3. PFD migliora l&#39;imaging confocale in foglie Immagini che mostrano LSCM cytoplasmically localizzato Venere fluorescenza (verde) e autofluorescenza della clorofilla (rosso) in A. thaliana lascia intatto ripreso in aria, acqua o PFD. Lunghezza d&#39;onda di eccitazione è 514 nm ed emissione di fluorescenza è stato registrato a 518 nm 604 nm per Venere e at 657 nm679 nm per cloroplasti. Ogni pannello è un&#39;unica sezione confocale preso da una pila di Z-11 immagini acquisite confocale a intervalli di 5 micron. Questi sono stati estratti da un pieno Z-stack di 59 immagini acquisite ad intervalli di 1 micron, che sono stati usati per produrre film supplementari. Misure di profondità sono date relativamente alla superficie epidermica. Immagini rappresentative, che sono stati trattati in modo identico, vengono visualizzati. Dettagli sperimentali come le impostazioni microscopio sono identiche a quelle trovate in Littlejohn et al., 2010 2. Scalebars rappresentano il 20 micron.

<ol>
	<li>Inoue, S., Pawley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foundations+of+Confocal+Scanned+Imaging+in+Light+Microscopy.">Foundations of Confocal Scanned Imaging in Light Microscopy.</a> Handbook of Biological Confocal Microscopy, 3rd. , 1-16 (2006).</li><li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. m. i. r. n. o. f. f., N, ., Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues.">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li><li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+perfluorinated+liquids.+Fed.">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li><li>Sch&#246;nherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+stomata+by+liquids.">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li><li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation.">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li><li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+ventilation+in+premature+lambs:+uptake,+biodistribution+and+elimination+of+perfluorodecalin+liquid.">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li><li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <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+perfluorocarbon+liquids+in+vitreoretinal+surgery.">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li><li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+blood:+synthetic+substitutes+from+fluorinated+compounds.">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li><li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon.">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li><li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorocarbons+to+study+freezing+in+plant+tissues.">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li><li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorophyll+fluorescence:+a+probe+of+photosynthesis+in+vivo.">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Questa è una tecnica semplice e facile da usare per migliorare la microscopia di aria riempita o otticamente complessi tessuti. Abbiamo dimostrato che la tecnica presenta alcuni vantaggi forte e speriamo che possa essere utilizzato per chiarire questioni biologiche riguardanti l&#39;aria ricca di tessuti. Per esempio sarebbe una scelta naturale per gli studi di attacco patogeno in mesofillo pianta o nei polmoni. Siamo anche consapevoli dei limiti della tecnica. Stiamo lavorando su una migliore corrispondenza indice di rifrazione, l&#39;uso con altri modi di microscopia, e la ricarica perfluorocarburo montaggio durante lunghi esperimenti tempi. Siamo anche consapevoli, però, che uno dei vantaggi principali di PFC, cioè di essere biologicamente inerte è uno rovescio della medaglia. PFC non si dissolvono rapidamente molecole biologiche che implica anche che non possono essere facilmente usati per fornire composti di interesse come gli ormoni, farmaci e altre piccole molecole o ioni.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Perfluorodecalin </td><td> F2 chimici </td><td> N / A </td><td> Telefono per ordinare. </td></tr><tr><td> Carolina di osservazione Gel </td><td> Lame biologica </td><td> 13-2700 </td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

Il problema di acquisizione di immagini ad alta risoluzione in profondità in campioni biologici è ampiamente riconosciuto 1. In aria piena di tessuti come il mesofillo spugnoso di foglie delle piante o dei polmoni dei vertebrati ulteriori difficoltà derivano da transizioni multiple in indice di rifrazione tra i componenti cellulari, tra le celle e spazi aerei e tra il tessuto biologico e il resto del sistema ottico. Inoltre, discordanze indice di rifrazione portare ad attenuazione di eccitazione fluoroforo ed emissione del segnale in microscopia a fluorescenza. Descriviamo qui l&#39;applicazione della perfluorocarburo, perfluorodecalin (PFD), come mezzo di imaging ottico infiltrativa che migliora la microscopia confocale a scansione laser (LSCM) immagini campione in profondità, senza ricorrere ad aumenti di danneggiare in potenza del laser e ha un minimo impatto fisiologico 2. Noi descriviamo il protocollo per l&#39;utilizzo del PFD con il tessuto foglia Arabidopsis thaliana, che è otticamente più complesse a causa della suastruttura (Figura 1). PFD ha una serie di attributi che lo rendono adatto per questo uso 3. L&#39;indice di rifrazione di PFD (1,313) è paragonabile a quella dell&#39;acqua (1.333) ed è più vicina a quella del citosol (circa 1,4) rispetto all&#39;aria (1.000). Inoltre, PFD è prontamente disponibile, non fluorescente e non è tossico. La bassa tensione superficiale di PFD (19 dyne cm -1) è inferiore a quella dell&#39;acqua (72 dyne cm -1) e anche al di sotto del limite (25 - 30 dyne cm -1) per la penetrazione stomatica 4, che permette di inondare gli spazi aerei mesofillo spugnoso senza l&#39;applicazione di un vuoto potenzialmente distruttivi o tensioattivo. Infine e soprattutto, PFD ha una grande capacità di dissoluzione di CO 2 ed O 2, che consente lo scambio di gas deve essere mantenuta nel tessuto invaso, minimizzando così l&#39;impatto fisiologico sul campione. Queste proprietà sono state utilizzate in varie applicazioni che includono parziale respiro liquido e del polmone inflazionezione 5,6, chirurgia 7, sangue artificiale 8, ossigenazione della crescita dei media 9, e gli studi di formazione di cristalli di ghiaccio nelle piante 10. Attualmente, è comune a montare il tessuto in acqua o in tampone acquoso per vivere l&#39;imaging confocale. Riteniamo che l&#39;uso di PFD come un mezzo di montaggio rappresenta un miglioramento rispetto alla pratica esistente e consente la semplice preparazione di live campioni foglia intera per l&#39;imaging.

Watch this Scientific Journal Video about A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium at JoVE.com

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Descriviamo l&#39;uso di perfluorodecalin come mezzo infiltrante di montaggio. Questo è un metodo semplice per migliorare la profondità delle immagini in<em&gt; Arabidopsis thaliana</em&gt; Tessuto di foglie con il minimo impatto fisiologico.

Un metodo semplice per l&#39;imaging Arabidopsis Foglie Utilizzando Perfluorodecalin come Media Imaging infiltrativa

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

a-simple-method-for-imaging-arabidopsis-leaves-using-perfluorodecalin

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

21.5K Views.  The University of Exeter. Descriviamo l'uso di perfluorodecalin come mezzo infiltrante di montaggio. Questo è un metodo semplice per migliorare la profondità delle immagini in Arabidopsis thaliana Tessuto di foglie con il minimo impatto fisiologico.

Video: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Vivere Confocale Imaging di sviluppare Arabidopsis Fiori

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Ricerca

Biologia dello sviluppo

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Una Camera Semplice per l&#39;Imaging Confocale a lungo termine dello sviluppo di radici e ipocotili

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Movimento shootward di CFDA Tracer caricato nei tessuti lavello inferiore di Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

Ambiente

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.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in 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 ...

Biologia

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Valutazione dei comportamenti fotosintetici mediante misurazioni simultanee della riflettanza delle foglie e delle analisi della fluorescenza della clorofilla

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Bioingegneria

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza activated cell sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

A lungo termine, ad alta risoluzione confocale Lapse Imaging Tempo di<em&gt; Cotyledon Arabidopsis</emEpidermide&gt; durante la germinazione

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Misurazione spaziale e temporale Ca<sup&gt; 2 +</sup&gt; Segnali in Arabidopsis Piante

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Registrazione in tempo reale In Vivo di Arabidopsis segnali di calcio durante l'alimentazione dell'insetto utilizzando un biosensore fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Un metodo Versatile per il montaggio di Arabidopsis lascia per l'Imaging di time-lapse videomicroscopia

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Un metodo semplice per l'imaging Arabidopsis Foglie Utilizzando Perfluorodecalin come Media Imaging infiltrativa

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Una Camera Semplice per l'Imaging Confocale a lungo termine dello sviluppo di radici e ipocotili

Movimento shootward di CFDA Tracer caricato nei tessuti lavello inferiore di Arabidopsis

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in Arabidopsis thaliana

Valutazione dei comportamenti fotosintetici mediante misurazioni simultanee della riflettanza delle foglie e delle analisi della fluorescenza della clorofilla

Cella di analisi specifica di

A lungo termine, ad alta risoluzione confocale Lapse Imaging Tempo di

Misurazione spaziale e temporale Ca

Registrazione in tempo reale In Vivo di Arabidopsis segnali di calcio durante l'alimentazione dell'insetto utilizzando un biosensore fluorescente

Un metodo Versatile per il montaggio di Arabidopsis lascia per l'Imaging di time-lapse videomicroscopia