IEM finansman Danimarka Araştırma Konseyi (FTP ve FNU) kabul etmek istiyorum. ERL bir ARC DP hibe desteği kabul etmektedir. AB Bitki hücre duvarları hibe Mükemmellik ARC Merkezi&#39;nin desteği ile hazırlanmıştır.

1. Doku Toplama ve Hazırlama <ol><li> Ilgi her doku için en az üç nüsha halinde bitki dokularının 100 mg taze ağırlık (10 mg kuru ağırlığının minimum) toplayın. Aşağıdaki adımları vejetatif doku hücre duvarı malzemesinin hazırlanması açıklanmaktadır. Depolama dokularının durumunda, un istenen nişasta enzimatik olarak daha önce 13 açıklandığı gibi hücre duvarı polimerlerin ekstraksiyon işlemine devam etmeden önce kaldırılır. </li><li> 24 boru adaptörü kümeleri ve 3 mm tungsten karbid boncuklar (30 Hz, 2 x 30 saniye) ile, Qiagen TissueLyser II kullanılarak sıvı azot ile birlikte ince toz haline örnekleri homojenize edilir. Sadece birkaç örnek işleniyor Alternatif olarak, eğer bir havanda kullanılır. </li><li> 10 ml plastik konik tüpler içine homojenatlarında aktarın. </li><li> Oda sıcaklığında 10 ml% 80 v / v etanol içinde homojenat yıkama ve 2 dakika boyunca kuvvetli bir şekilde vorteks hücre çeperi materyali hazırlanır. </li><li> 3.500 x g&#39;de santrifüjleyin ve supernatant atın. Süpernatant berrak olana kadar% 70 etanol, özellikle, klorofil içeren dokular için, ya da en az üç kez yıkama tekrarlayın. </li><li> % 100 aseton ile son yıkama yapın ve gecede kurumasını bekleyin Alkol Çözünmeyen Kalıntı (AIR) içeren granül bırakın. </li><li> Elek AIR ince, homojen toz elde etmek ve kötü, bazen homojenat kalır zemin partikülleri büyük kaldırmak için 0.4 mm 2 mesh ile örnekleri. </li><li> Mikrotüpler, numunelerin karışım yardım etmek için bir 3 mm cam boncuk içeren her birine 10 mg AIR örnek tartılır. </li></ol> 2. Hücre Duvarı glukanlardır çıkarımı <ol><li> Pektinler ile ilişkili pektinler, ve polimer her bir örnek için 50 uM CDtA (pH 7.5), 500 ul ilave edilerek numune çıkarılır. </li><li> Kısaca girdap boruları çözücü numune malzeme ile temas halinde olmasını sağlamak ve böylece 8 Hz, 3 saat boyunca TissueLyser ile karıştırılır. </li><li> Dikkatle, 12.000 xg&#39;de santrifüjleyin r4 ° C&#39;de süpernatan ve mağaza kaydının silinmesi </li><li> 13,000 x g hızında kalan herhangi bir çözücü, girdap ve santrifüj sulandırmak ve çıkarmak için de-iyonize su, 1 ml granül yıkayın. Pelet bozmadan bu adımı yineleyin ve sonraki adıma geçmeden önce tüplerin tüm sıvı kaldırmak. </li><li> Çapraz bağlama glukanlardır ardışık olarak% 0.1 ile 4M NaOH ile 500 ul, kalan hücre duvarı peleti elde edilir adımlar 2.1 &#39;de tarif edilen prosedür kullanılarak w / v NaBH4, - CDtA çıkarılması için 2.4. NaBH4 böylece polisakaritlerin soyulma baz engelleyen bir alkol ile polisakaritler azaltılması sonunda aldehit (ya da keto) grubu azaltmak için eklenir. </li><li> Santrifüjlemeden sonra, süpernatan tekrar çıkarılır, 4 ° C de depolanır ve peletlerin bir sonraki ekstraksiyon işlemine geçmeden önce de-iyonize su ile iki kere yıkandı. </li><li> Isteğe bağlı bir adım olarak, bu tür selüloz gibi polimerlerin kalıntı, 500 ul cadoxen (% 31 v / v 1,2-diami ile ekstre edilmiştir2.4 - 2.1 adımlar açıklandığı gibi aynı prosedürü kullanarak 0.78 M CdO ile noethane). Alternatif olarak, kalan pelet mutlak selülozik içeriği Asetik / Nitrik testler (Tartışma bakınız) kullanılarak tespit edilebilir. </li></ol> 3. Baskı Mikroarray&#39;ler <ol><li> Santrifüj süpernatantlar herhangi bir partikül madde kaldırmak için 13.000 xg&#39;de hücre duvarı polimerleri ekstre içeren. </li><li> Bir de polipropilen 384 numune doku tipi ve ekstraksiyon tipine göre düzenlenir Önceden tasarlanmış özel bir düzen kullanarak mikrotiter plaka içine her bir örnek 50 ul yükleyin. </li><li> Deiyonize su ile 0, 5 ve 25 × seri dilüsyon serisi hücre duvarı polimer örneği seyreltin. </li><li> Böyle pin yüksekliği, toplama ve bekleme süresi, ve yıkama adımları gibi parametreler microarrayer kontrol yazılımı ayarlanır. </li><li> Baskı bölmesinin nem örnek buharlaşmasını önlemek için% 60 kontrol edilir. </li><li> Yazdırma işi LabNEXT softwa ile başlatıldığıyeniden ve mikroarray düzeni karşılık gelen bir program. </li><li> Robot makine düz bir levha üstüne tutturulmuş 20 x 20 cm nitroselüloz membran üzerinde numune plaka çözümleri yazdırmak için kılcal kanal işaretçilerine kullanır. Dizisindeki her nokta çözümü 15 nl içerir ve üç nüsha halinde basılır. </li><li> Yazdırma işi tamamlandıktan sonra Özdeş mikroarrayler Tek tek dizileri içine membran ve kesim birbirlerine yanında yazdırılır. </li><li> Her bir deneyde, diziler daha fazla ya da daha az örnekleri, seyreltici ya da çoğaltır yerleştirmek için modifiye edilebilir. </li></ol> 4. Glukanıdır Mikroarray&#39;ler arasında Sondalama <ol><li> Baskıdan sonra, spesifik olmayan bağlayıcı azaltmak için fosfat içinde çözüldü ağırlık / hacim kaymağı alınmış süt tozu 2 saat boyunca oda sıcaklığında (Mpbs) tamponlu tuzlu su içinde% 5 bireysel mikrodizileri bloke eder. </li><li> Mpbs içinde 2 saat için hücre-duvar glikan epitoplar için spesifik monoklonal antikorlar ile Sondası mikrodizileri. Monoklonal antib çoğunluğuHücre duvarı glukanlardır karşı odies üç firmalardan ticari olarak temin edilebilirler; Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ), Carbosource Hizmetleri ( <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ) ve PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ). </li><li> Bir negatif kontrol, sadece Mpbs ve birincil antikor ile birlikte inkübe bir mikro içerir. </li><li> Fosfat içinde 3 kez spesifik olmayan bağlanma kaldırmak için 5 dakika için (PBS), tamponlu tuzlu su mikrodizileri yıkayın. </li><li> 2 saat için Mpbs içinde yabanturpu peroksidaz (HRP) konjuge sekonder antikor ile mikrodizileri kontrol edin. Hücre duvarı glukanlardır karşı en monoklonal antikorlar, anti-fare veya anti-sıçan sekonder antikor gerektirir. </li><li> Spesifik olmayan bağlanma kaldırmak için 5 dakika için 3 kez PBS tamponu ile yıkama adımları tekrarlayın. </li><li> Kromojenik (3,3-diaminobenzidin) ya da chemil kullanılarak mikrodizileri geliştirmekuminecent (luminol) yüzeyler. </li></ol> 5. Niceleme <ol><li> Kalkınma sonra, yüksek çözünürlüklü (1200 dpi) masaüstü tarayıcı kullanarak bireysel mikroarrayler taramak ve negatif, 16 bit TIFF dosyaları (Şekil 3) olarak kaydedin. </li><li> Otomatik ızgara aracı ile donatılmış Görüntü İşleme Yazılımları (LabNEXT) Xplore kullanarak her nokta ayrılmaz yoğunluğunu hesaplayın. Tamamlayıcı nokta yoğunluğu her bir nokta çevreleyen ızgara alanı içinde piksellerin toplamı elde edilir. </li><li> Her mikroarray için grid veri bir txt dosyası olarak ihraç edilir ve manuel analizi için bir Excel elektronik tablo içine alınabilir. Bir çevrimiçi araç ( <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/</a> ) otomatik olarak tercüme ve bireysel txt dosyalarından veri işlemek için geliştirilmiştir. </li><li> İntegral nokta yoğunluğu baskı çoğaltır genelinde ortalama bir &#39;ortalama bir nokta elde etmek dilüsyonları edilirHer numune için yoğunluk &#39;değeri (Şekil 2). Alternatif olarak, bir dizi üzerinde sadece tek bir seyreltme değerine karşılık gelen nokta sinyaller her bir numune için göreli bolluk glikan epitop ölçmek için kullanılır. </li><li> Farklı numuneler arasındaki göreceli ortalama nokta yoğunlukları excel veya çevrimiçi heatmapper araçları (koşullu biçimlendirme kullanarak bir heatmap (Şekil 4) olarak sunulmaktadır <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ). Her antikor tipi için veriler 100 düzeltilir ve% 5 eşik değeri arka sinyal ve yanlış pozitif kaldırmak için uygulanmaktadır. </li></ol>

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Çıkar çatışması ilan etti.

CoMPP birkaç gün içinde yüzlerce bitkisel kaynaklı numunelerin glukanıdır kompozisyonu profilleme için hızlı ve hassas bir yöntemdir. Bu yöntem, lektinler, reseptörleri ve antikorları 16 gibi glukanıdır-bağlayıcı proteinler ile karbohidrat etkileşimleri yüksek verimli tarama zaten mevcut bakteriyel veya memeli glukanıdır dizi platformlar tamamlar. Hücre duvarı glukanlardır tespit etmek için kullanılabilir probları geniş bir çeşitliliği ile, bitki hücre duvarı 8,9 ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> 3 mm Tungsten Karbür boncuk </td><td> Qiagen </td><td> 69997 </td><td></td></tr><tr><td> Koleksiyon mikrotüpler (1,2 mm) </td><td> Qiagen </td><td> 19560 </td><td> 1.5 ml mikrofüj tüpler de kullanılabilir </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85300 </td><td></td></tr><tr><td> 3 mm cam boncuklar </td><td> Sigma Aldrich </td><td> Z143928 </td><td></td></tr><tr><td> CDtA </td><td> Sigma Aldrich </td><td> 34588 </td><td></td></tr><tr><td> Oksit Kadmiyum </td><td> Sigma Aldrich </td><td> 202894 </td><td></td></tr><tr><td> 1,2-diaminoetan </td><td> Sigma Aldrich </td><td> 03550 </td><td></td></<html> tr&gt; <tr><td> Nitroselüloz membranı (0.22 mikron gözenek boyutu) </td><td> GE-su ve proses teknolojileri </td><td> EP2HY00010 </td><td> farklı gözenek boyutlu membranlar farklı pin tipleri için uygundur </td></tr><tr><td> Xact II microarrayer robot </td><td> Labnext </td><td> 001A </td><td> Xact II robot nitroselüloz membran bağlı olduğu özel bir 20 x 20 cm seramik plaka takıldı </td></tr><tr><td> RM mikroarray işaretçilerine Xtend </td><td> Labnext </td><td> 0037-350 </td><td> işaretçilerine zarlarında tespit için uygun olmalıdır </td></tr><tr><td> 384 oyuklu plakalar (polipropilen) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> Anti-glikan monoklonal antikor </td><td> / Bitki Probları CarboSource / Biosupplies </td><td></td><td> Web siteleri; PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ), Carbosource (ww.carbosource.net &quot;target =&quot; _blank &quot;&gt; www.carbosource.net) ve Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> Anti-sıçan IgG (tüm molekül) - keçi üretilen peroksidaz antikor. </td><td> Sigma </td><td> A9037 </td><td> İkincil antikor tipi (örneğin fare, sıçan, keçi vb yükseltilmiş) kullanılan birincil antikor bağlıdır. </td></tr><tr><td> SIGMA FAST 3,3 &#39;-diaminobenzidin tablet </td><td> Sigma </td><td> D4293 </td><td> reaktif geliştirilmesi tipi, kullanılan ikincil antikorlar ve tespit yöntemi (colourmetric, ya da chemiluminecent) bağlıdır. </td></tr><tr><td> SuperSignal West Pico Chemiluminescent Yüzey </td><td> Thermoscientific </td><td> 34080 </td><td> yukarıya bakın </td></tr><tr><td> Görüntü İşleme Yazılımları Xplore </td><td> LabNext </td><td> 008 </td><td> Otomatik gridding araçları ile birçok yazılım türleri mevcutturmikroarray noktalar piksel değerini ölçmek için. </td></tr><tr><td> Bitki polisakkaritler </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Bitki hücre duvarları fizyolojisi ve bitki gelişmesinde önemli bir rol oynar ve insan topluluklarının (örneğin tahta, kağıt, tekstil ve biyoyakıt endüstrisi) 1,2 için hammadde sağlamak heterojen glukanlardır karmaşık matrisler vardır. Bununla birlikte, bu bileşenlerin biyosentezini ve işlevlerini anlamada zorlu kalır. Hücre duvarı glukanlardır kimyasal ve yapı taşları, glikozil kalıntılarının karmaşıklığı nedeniyle conformationally çeşitlidir. Bu formu çoklu pozisyonlarda bağlar ve halka yapısı farklı, izomerik ya da anomerik konfigürasyon, ve ek olarak, non-şeker artıkları bir dizi ile sübstitüe edilmiştir. Glukanıdır kompozisyonu hatta farklı hücre ve / veya doku tipleri veya tek bir hücre duvarı 3 alt alan adları değişir. Ayrıca, bunların bileşimi, aynı zamanda 1 geliştirme sırasında, ya da çevresel işaret 4 yanıt olarak modifiye edilir. Ex2.000 genlerin cess Bitki hücre duvarlarının heterojen glukanlardır karmaşık matrisler Arabidopsis 5 hücre duvarı glukanıdır biyosentezi ve modifikasyonu yer alacağı öngörülmüştür olanlarıdır. Bununla birlikte, biyosentez genlerinin göreceli olarak az sayıda işlevsel olarak 4,5 karakterize edilmiştir. Genlerin çoğu zaman farklı olarak hücre tipleri 6 arasında, sık sık düşük seviyelerde ifade edilir çünkü karşıt genetik yaklaşım zordur. Ayrıca, mutant çalışmalar genellikle uygun hücre duvarı işlevi 7 korunmasını sağlamak için gen fazlalık veya telafi edici mekanizmalar tarafından engellenmiştir. Böylece yeni yaklaşımları hızla glukanıdır yapıların çeşitli karakterize etmek ve anlayış hücre duvarı biyosentezi ve modifikasyonu fonksiyonel genomik yaklaşımlar kolaylaştırmak için ihtiyaç vardır. Monoklonal antikor (mAb) 8,9 Bitkilerde glukanıdır yapısı ve dağılımı belirlenmesi için önemli bir araç olarak ortaya çıkmıştır. Bu dist tanımakpektinler, xyloglucans, xylans, mannans, glukan ve arabinogalactans gibi bitki hücre duvarının glukanlardır ana sınıfları, içinde bulunan inct epitoplar. Son zamanlarda bunların kullanımı bitki ve doku tipleri eşzamanlı 9,10,11 geniş bir yelpazede glukanlardır göreli bolluğu belirlemek için geniş çaplı tarama deneyler kadar uzatılmıştır. Burada düşük reaktif ve numune hacimleri ile minyatür bir mikroarray platformu kullanarak ekranlı olması (100 sn) çoklu örnekleri sağlar Kapsamlı Mikroarray Polimer Profiling (CoMPP) (Şekil 1 ve 2) 10,11 adlı bir mikroarray tabanlı glukanıdır tarama yöntemi sunuyoruz. Mikroarray üzerinde nokta işaretleri resmen glukanıdır epitop oluşumu hakkında yarı-nicel veri vermek için ölçülebilir. Bu yaklaşım karmaşık biyolojik sistemlerin 12 glukanıdır değişiklikleri izleme ve hücre duvarı kompozisyonları küresel bir bakış sağlamak için uygundur, özellikle önceki bilgilerif Bu kullanılamıyor.

Nicotiana alata çiçeklerden altı doku tipleri (anter filamentler, polen, yumurtalıklar, yaprakları, sepals ve stigma) olarak glukanlardır göreli bolluğu CoMPP kullanılarak belirlendi. Şekil 3A kısmen (düşük) methylesterified homogalacturonan (HG), pektik polisakkaritler 14 oluşur bir epitopu için mAb JIM5 özel ile probed edilmiş bir temsilcisi mikroarray gösterir. JIM5 epitopunu çiçek dokuların CDtA özleri algılanırsa ancak mercek dokusu (Şekil 3A ve 4A) pol...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Denilen bir tekniği<strong&gt; C</strong&gt; Omprehensive<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strongBitki hücre çeperi glukanlardır karakterizasyonu için&gt; olymer profil (CoMPP) tarif edilir. Bu yöntem, biyolojik bağlamlarda geniş bir aralıkta glikan geçtiği tarama izin veren bir minyatür mikrodizi analitik platformu ile tanımlanan glikan-epitoplara karşı yönelik monoklonal antikorlar özgüllük birleştirir.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

glycan-profiling-of-plant-cell-wall-polymers-using-microarrays

Research

JoVE Journal

Biology

14.5K Görüntüleme.  University of Melbourne. Denilen bir tekniği C Omprehensive M Icroarray P olymer profil (CoMPP) tarif edilir. Bu yöntem, biyolojik bağlamlarda geniş bir aralıkta glikan geçtiği tarama izin veren bir minyatür mikrodizi analitik platformu ile tanımlanan glikan-epitoplara karşı yönelik monoklonal antikorlar özgüllük birleştirir.

Video: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

Bakteriyel Gen İfadesi Analizi Mikroarray&#39;ler Kullanımı

Biyoloji

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Asidik pH ve Alpha-Expansin İndüklenmiş Ölçüm Bitki Hücre Duvarı Uzantısı (Creep)

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

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.

Hücre tipi Özgül gösterilmeden FACS yardımlı kullanarak Doku ve Organ spesifik Fitokrom Yanıtları incelenmesi 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 ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Mikroarray&#39;ler kullanarak tRNA Aminoacylation (Şarj) Seviyeler genom Analizi

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Ekstrasellüler polisakkaritler Oligo Kitle Profil (Olimp)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

İzotop Etiketleme kütle spektrometresi kullanarak Tiyol Redoks Proteom profil

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histokimyasal boyama<em&gt; Arabidopsis thaliana</em&gt; Ortaöğretim Hücre Duvar Elemanları

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

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

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

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

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

Kristalin Selüloz Birikimin Yüksek Çözünürlüklü Niceleme<em&gt; Arabidopsis</em&gt; Kökler Doku spesifik Hücre Duvarı Değişiklikler Monitör

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

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Bireysel Hücre Tiplerinin transkripsiyonel Profiling için bir alet olarak lazer destekli mikrodiseksiyon (LAM)

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Bitki Polizom Profiling için bir Kolay Yöntem

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...