Bitkilerde Fitokrom yanıtları Montgomery laboratuarında, Ulusal Bilim Vakfı (hibe BLM hiçbir MCB-0.919.100) ve Kimya Bilimleri, Yerbilimleri ve Biyobilimler Bölümü, Temel Enerjisi Bilimler Bürosu, Fen Dairesi, ABD Dışişleri Bakanlığı tarafından desteklenen Enerji (BLM hibe hiçbir DE FG02 91ER20021). Biz çekimler sırasında Melissa Whitaker teknik yardım için teşekkür ederim ve eleştirel deneysel yardım, gelişmekte olan ve konfokal yardım için Arabidopsis protoplast sıralama ve Dr. Melinda Çerçeve protokoller Sıralama Floresan-Aktif Hücre optimize yardım için Dr Louis King, Stephanie Costigan yazının okuma mikroskopi. Marlene Cameron, grafik tasarım yardım ve editoryal yardım için Karen Kuş için teşekkür ederiz.

1. Bitki Büyüme <ol><li> Izole Onaylandı UAS-BVR X GAL4-GFP arttırıcı tuzak hattı olarak 4 açıklanan (özeti için bkz: Şekil 1) ve vahşi-tip veya ebeveyn hatları toprağa ekilir, yani satır başına ~ 2000 sterilize tohumları. </li><li> Bitkiler 22 100 μmolm -2 s -1 beyaz aydınlatma altında toprak 5 hafta boyunca yetiştirilen ° C ve% 70 nem. </li></ol> 2. Yaprak Protoplast İzolasyonu (Denecke ve Vitale 9 uyarlanmış) <ol><li> Protoplast izole etmek için, TEX tampon hazırlamak. (N-morfolino) ethanesulfonic asit (MES, 2.56 mM), 0.75 gr kalsiyum klorür dihidrat (CaCl 2 .2 H 2 O, 6.75 - 3.1 g Gamborg Kullanıcı B5 tuzları, 0.5 g 2: 1 litre TEX tampon için, aşağıdaki bileşenleri dışarı tartmak mM), 0.25 g amonyum nitrat (NH 4 NO 3, 3.12 mM), 136.9 g sakaroz (0.4M). deiyonize, distile su ~ 900 ml (GKD 2 O) tamamen bileşenleri çözülür ve 1 ile 5.7 pH getirmek M KOH. 1 litre ve bir vakum pompasına bağlı bir şişe top 0.2 mikron filtre ile sterilize filtre son hacim kazandırın. </li><li> Bitkilerin yeşil, sağlıklı yaprakları (gevşek bir beherdeki dolu yaprakları ~ 250 ml) toplayın ve ~ 40 ml GKD 2 O 4 kez ile yıkayın, iki kez steril GKD 2 O. ile durulama # 20 bistüri, kesilmiş yapraklar ince şeritler halinde ve eşit iki adet 50 ml steril plastik tüpler içine doku bölmek. 1x 10x yaprak sindirim stok solüsyonu (10x yaprak sindirim stok solüsyonu 5 ml kısım ekleyerek yaprak sindirim karışım hazırlayın: w / v Macerozyme R-10,% 4 w / v TEX tampon selülaz &quot;Onozuka&quot; R-10,% 2 çözülür 45 ml taze TEX tampon 0.2 mm) -80 ° C&#39;de 5 ml alikotları filtre ve dondurma ile sterilize edin filtre. ~ 25 ml 1x tüm yaprak doku kapsayacak şekilde 50 ml tüp başına yaprak sindirim karışımı ekleyin. </li><li> Vakum, nazik sallayarak bir rocker oda sıcaklığında 3 saat inkübasyon takip bir su pompası bağlı bir vakum desikatörde oda sıcaklığında 1 saat süreyle açık tüpler sindirim karışımı yaprak dokusu sızmak. 1x yaprak sindirim karışımı yaprak dokusu ile Kapaklı tüpler Işığa maruz kalmasını önlemek için bu işlem sırasında alüminyum folyo ile sarılı tutulur. </li><li> 3 saat sonra, yaklaşık 2 dakika protoplast serbest için rocker hızını artırmak. Pislikleri temizleyin ve steril bir cam beher süzüntü toplamak için iki kat steril peynir bez ile ham protoplast süspansiyon Filtre. Steril bir Petri kabı içine steril 100 mikron naylon örgü ile süzüntü Filtresi. Üzerinden akış toplayın ve yeni bir steril 50 ml tüp aktarmak. Steril Petri bulaşık yıkama ve yüzeye yapışan herhangi bir protoplast toplamak için 15-20 ml taze TEX tampon kullanın. </li><li> 10 100xg bir salıncak kova rotor kullanarak ° C için 15 dakika (6 Hızlanma, Yavaşlama 0) üzerinden akış santrifüjleyin. </li><li> ~ 25 Kaldır - 30 ml kayan protoplast katman, altında kalan ve pelet enkaz içeren sıvı, kayan protoplast tabakası rahatsız edici ve ~ 10 bırakarak peristaltik bir pompa bağlı steril 9 &quot;cam Pasteur pipeti ile 15 ml. </li><li> Yavaşça protoplast resuspending son hacim 40 ml taze TEX tampon ekleyin. </li><li> Mümkün olduğunca çok hücresel enkaz kaldırmak için adımlar 2,5-2,7 iki kez tekrarlayın. Santrifüj zaman, ilk tekrarından 10 dakika ve son tekrarından 5 dakikaya kadar azalır. </li><li> Kayan protoplast yeni bir 15 ml&#39;lik tüp içine bir kesim, steril 1 ml transfer pipet ile aspire edin. 4 az 2 saat kadar sıralama ya da karanlıkta saklayın ° C sıralama kadar doğrudan geçin. </li></ol> 3. Floresan-Aktif Hücre Ayırma (FACS) Protoplast Sıralama <ol><li> Sıralamadan önce, protoplast bütünlüğü ve protoplast havuzu GFP floresan varlığını teyit etmek için bir uyarma için 488 nm lazer kullanarak Konfokal Lazer Tarama Mikroskobu (CLSM) tarafından protoplast inceleyin. Minimal enkaz FACS sıralama memesi tıkanmasını önlemek için gerekli. </li><li> FACS üzerinden TEX tampon (BD FACSVantage SE, BD Biosciences) yaklaşık 9 psi uyarlanmış protokolü 10 aşağıdaki bir sistem basıncı ile 200 mikron memesi kullanarak, 6.000 ve 15.000 arasında olay oranları bir makro sıralama kafasına göre izole protoplast. </li><li> Wild-tip olmayan GFP protoplast otofloresans eşikleri belirlemek için kullanılır. </li><li> GFP pozitif protoplast, GFP floresan 530/30 bir bant kullanarak tanımlamak için bir 488-nm argon hattı kullanarak bir hava soğutmalı argon lazer (Spectra Fizik Model 177, Newport Corporation, Irvine, CA) 100 mW işletilen tür hücreleri toplamak için geçiş filtresi. </li><li> GFP pozitif protoplast toplanmasını takiben, adım 3.1 &#39;de ayrıntılı olarak CLSM göre sıralanmış protoplast inceleyin. </li><li> Qiagen RNeasy Bitki Mini Kit kullanılarak sıralanır protoplast toplam RNA ayıklayın. Affymetrix GeneChip İfade açıklandığı gibi cDNA sonra hibridizasyon için hazırlanmış olabiliression Analizi Teknik Manuel ve sonra AtH1 Arabidopsis Affymetrix tüm genom dizileri melezleşmiştir. </li></ol> Temsilcisi Sonuçlar Biz FACS (Şekil 2) GFP kanal GFP pozitif hücrelerin önemli bir numarası algıladı. Vasküler ve dermal ifade ile çizgi, J1071, ~% 1.4, GFP pozitif protoplast sahipken, GFP arttırıcı-trap ebeveynler, yapısal GFP ifade doğrultusunda, J0571 görüntülenir ~% 17 -% 24, GFP pozitif protoplast kullanarak optimizasyon tayinlerde (Tablo 1). 1 saat J0571 protoplast 3 x 500 ul (~ 1.5 ml toplam protoplast süspansiyon) sıralama ~ 100.000 GFP pozitif protoplast verdi. 1,5 saat boyunca J1071 protoplast 3 x 500 ul (~ 1.5 ml toplam protoplast süspansiyon) sıralama ~ 3000 GFP pozitif protoplast verdi. Sıralama (Şekil 3C ve 3E) gerçekleştirildi önce Konfokal görüntüleri hem J0571 ve J1071 örnekleri için çok yüksek bir verim protoplast gösterilir ve sıralaması fraksiyonları tek parlak GFP fluoresan protoplast (Şekil 3G ve veri gösterilmemiştir) yer. Bu bulgu, bu sağlam GFP pozitif protoplast FACS üzerinden düzenlenmiş olabilir onaylar. Olmayan GFP protoplast de sıralanır ve ayrı bir kanal alınabilir. GFP olmayan protoplast holophytochrome düzeylerinin azaltılması üzerinde gen ekspresyonu belirli değişiklikler tespit sonraki mikroarray analizleri için ideal bir negatif kontrol olarak hizmet vermektedir. Izole protoplast (1 ml) fluorospectrometry (Şekil 4) tespiti için yeterli miktarda verim RNA RNA ekstraksiyonu. İzole RNA NanoDrop bir alet kullanarak (NanoDrop 1000, Thermo Scientific) NanoDrop 3.7.1 RNA ölçüm yazılımı tarafından değerlendirilir ve sayısal oldu. RNA verim C24 yabani tip protoplast veya FACS-sıralaması, GFP pozitif arttırıcı tuzak protoplast kullanmak için gerekli olan en az 20 ng aştı önceden sıralanmış RNA etiketleme mikroarray (Tablo 2) testleri. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Geliştirici Trap Line </td><td> % GFP pozitif protoplast </td><td> Sıralanmış GFP protoplast sayısı </td></tr><tr><td> J0571 </td><td> 17.18 ~% 24,06% </td><td> 26.400 ~ 36.000 </td></tr><tr><td> J1071 </td><td> ~% 1.43 </td><td> 1,000 ~ 1,300 </td></tr></tbody></table> Tablo 1. sıralama ve Floresan Aktif Hücre Sıralayıcısı (FACSVantage BD) ile 500 mcL protoplast süspansiyon çalışan tarafından toplanan GFP pozitif protoplast sayısı önce iki arttırıcı tuzak hatları GFP pozitif protoplast yüzdesi. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Bitki Hattı </td><td> RNA verimi (ng) </td></tr><tr><td> C24 WT </td><td> 12486,5 </td></tr><tr><td> J0571 </td><td> 60 </td></tr><tr><td> J1071 </td><td> 71,5 </td></tr></tbody></table> Tablo 2. protoplast RNA izolasyonu hesaplanmıştır. RNA iki arttırıcı tuzak hatları ön-C24 sıralaması yabani tip ya da sıralanmış GFP pozitif protoplast izole ve sayısal olarak ifade edildi. <img src="/files/ftp_upload/1925/1925fig1.jpg" alt="Şekil 1" /> Şekil 1 Biliverdin Redüktaz (BVR), transgenik Arabidopsis thaliana bitkilerin ifade GAL4 arttırıcı-trap tabanlı indüksiyon . (A). GAL4 duyarlı bir GFP işaretleyici gen içeren GAL4 tabanlı arttırıcı tuzak hatları, bir kütüphane seçilen bir kişinin hedef genin ifade ikna etmek için bir GAL4 duyarlı hedef geni içeren bir satır GAL4 içeren işaretlenmiş hücreler ile geçilebilir GFP floresan. Dr Jim Haseloff (http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html) bir rakam geçerli. (B) üst hatlarında Fitokrom kromofor, phytochromobilin (PΦB) ve holophytochrome üretimi. (C) Sol, biliverdin redüktaz (BVR) BR ve PΦR etkinlik biliverdin IXα (BV IXα) ve PΦB azaltılması, sırasıyla. BVR faaliyet sonuçları PΦB tükenmesi ve holophytochrome fotoaktif üretim azalmasına yol açar. Sağ, BVR tarafından katalizlenen reaksiyon gösterilir. <img src="/files/ftp_upload/1925/1925fig2.jpg" alt="Şekil 2" /> Şekil 2 Floresan Aktif Hücre Ayırma (FACS) satın alma nokta grafikleri. Protoplast hücre Karşılaştırma C24 vahşi tip (A), J0571 (B) ve J1071 (C) sıralama. (A) 488-nm argon lazer ile heyecanlandım ve otofloresans eşiğini belirlemek için kullanılan non-GFP floresan C24 tipi vahşi protoplast Toplama nokta arsa. (B) ve (C) satın alma nokta araziler, 488 nm lazer uyarma yanıt olumlu GFP protoplast oranlarda göstermektedir. B ve C sınırlarını Floresan-Aktif Hücre Sıralayıcısı göre sıralanır (FACSVantage BD) ve toplanmıştır GFP pozitif hedefler R3 sıralama kapıları. Kırmızı kanalı v gösterirprotoplast ve yeşil kanal klorofil otofloresans alues ​​GFP floresan için değerleri gösterir. <img src="/files/ftp_upload/1925/1925fig3.jpg" alt="Şekil 3" /> Şekil 3 hücre sıralama için kullanılan bitki protoplast mikroskopisi. Konfokal lazer Floresan-Aktif Hücre Sıralayıcısı (FACS) ile (G) sıralama önce (A, C, E) ve sonra protoplast, taranmış görüntüler. B, D, F ve H DIC görüntüleri. C24 vahşi tip (A, B), J1071 (C, D) ve J0571 (E, F, G, H) protoplast gösterilmiştir. Ve 488 nm lazer ile uyarma elde Otofloresans (LP 650 nm) - Görüntüler C, E ve G GFP floresans (575 nm BP 505 nm) görüntüler birleştirilir. H üzerinden 63x yağ altında 4 taramaları ortalama. Bar = 10 mm. <img src="/files/ftp_upload/1925/1925fig4.jpg" alt="Şekil 4" /> Şekil 4 fluorospectrometry tarafından protoplast izole edilen RNA Niceleme. RNA (A) C24 vahşi tip, (B) J0571, ve (C) J1071 ayıklanır. Öncesi sıralaması vahşi tip protoplast RNA gösterir. B ve C Floresan-Aktif Hücre Ayırma (FACS) göre sıralanır GFP pozitif protoplast RNA göstermektedir. RNA kantitasyonu yazılım, NanoDrop 3.7.1 (NanoDrop 1000, Thermo Scientific).

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Mikroarray&#39;ler yoluyla gen ekspresyonu (1) Arabidopsis fide genlerinin% 30 daha fazla ışık düzenlenir 11 olduğunu göstermiştir ve (2) Fitokrom sinyalizasyon kaskad 12, 13 yer hafif sinyal iletimi proteinleri kodlayan genlerin büyük bir grup belirlemiştir . Bu tür deneyler ışığın gen ekspresyonu hızlı ve uzun vadeli değişikliklere de neden olduğunu düşündürmektedir. Her havuz phytochromes gelişimsel ve adaptif yanıtlar yalnızca bir alt kümesini kontrol edebilir. Ayrıca, mansap sinyalizasyon bileşenler, hücre ve doku-spesifik bir şekilde 2, 14, 15 phytochromes ile etkileşim olasıdır . Downstream aday genlerin ifade olabilir yukarı veya aşağı-hedefli BVR ifadesi üzerine düzenlenir. UAS-BVR X GAL4 GFP döl ve ebeveyn hatları gen ekspresyonu değişiklikleri karşılaştırmalı analizi sayesinde, gen ekspresyonu belirgin değişiklikler lokalize Fitokrom eksikliği bu tür değişiklikler sonucu belirleyebilir. Gen ekspresyon profilleri dayanarak, biz Fitokrom aracılı hücre-hücre sinyal olumlu ve olumsuz faktörleri kodlayan genlerin tespit edebilir. Son veriler, birkaç yüz transkript 16 bitki doku protoplasting tarafından indüklenen olduğunu düşündürmektedir. Böylece, mikroarray analizi için ideal bir negatif kontrol RNA sıralama sırasında toplanan olmayan GFP protoplast izole edilmiştir. Bitki doku protoplasting tarafından indüklenen Transkriptler özellikle doku ve organ spesifik Fitokrom sinyalizasyon ve / veya yanıtların yer alan hedef genlerin belirlenmesi, bu denetimleri kullanarak veri analizi dışında olabilir. Hücre türüne özgü ifade profil üzerinden tanımlanan genlerin ifade kararlı-durum değişiklikleri onaylamak için, QRT-PCR sıralaması GFP-pozitif ve GFP-negatif protoplast Poli (A) RNA yürütülen olabilir. Ifade mikroarray analizleri önemli ölçüde değişti ve QRT-PCR ile teyit Genler ayrık Fitokrom aracılı hücrelerarası sinyalizasyon dahil aday olarak tanımlanır. T-DNA ekleme mutantlar aday genler zaten varsa, onlar ışık engelli fenotipleri için analiz edilebilir. Aday genler ve vahşi tip Fitokrom mutantlar bilinen mutasyonları taşıyan mutantlar fenotipleri karşılaştırarak, aday genler düzenleyici rol aldığı özel yanıtları tanımlayabilirsiniz. Aday gen mutasyonları ile mutantlar Fitokrom eksikliği fenotipleri ekran varsa, bu tespit downstream hedef genlerin farklı Fitokrom düzenlenmiş yanıtları arabuluculuk yer aldığını teyit edecektir.

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<td>Anti-BVR antibody</td>
<td>&nbsp;</td>
<td>QED Bioscience Inc.</td>
<td>56257-100</td>
<td>&nbsp;</td>
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<td>Cellulase &#x201C;Onozuka&#x201D; R-10</td>
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<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>MSPC 0930</td>
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<td>Gamborg&#x2019;s B5 basal salt mixture</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5768</td>
<td>&nbsp;</td>
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<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>PTC 001</td>
<td>&nbsp;</td>
<tr>
<td>MES, low moisture content</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
<td>&nbsp;</td>
<tr>
<td>Murashige and Skoog salts</td>
<td>&nbsp;</td>
<td>Caisson Laboratories</td>
<td>74904</td>
<td>&nbsp;</td>
<tr>
<td>Phytablend</td>
<td>&nbsp;</td>
<td>Caisson Laboratories</td>
<td>28302</td>
<td>&nbsp;</td>
<tr>
<td>RNeasy Plant Minikit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>16419</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

investigating tissue- and organ-specific phytochrome responses using facs-assisted cell-type specific expression profiling in arabidopsis thaliana

Işık aracılık gelişimsel ve adaptif süreçler bir bitkinin yaşam döngüsü boyunca bir dizi. Bitkiler kullanan ışık emici fotoreseptörlerin duygusu ve ışık uyum adı verilen moleküller. Uzak-kırmızı / kırmızı ışık emici Fitokrom fotoreseptörlerin yoğun çalışılan edilmiştir. Phytochromes çalışılmıştır olan tüm yüksek bitki sistemleri farklı ve birbiriyle çakışan fonksiyonları ile proteinlerin bir aile olarak var<sup&gt; 1</sup&gt;. Çiçeklenme ve tohum çimlenme yaşlanması yoluyla aralığı Fitokrom aracılı ışık tepkiler, genellikle belirli bitki doku veya organların lokalize<sup&gt; 2</sup&gt;. Mutasyon analizleri aracılığıyla, bireysel ve gereksiz Fitokrom fonksiyonlarının keşfi ve aydınlatılması rağmen, photoperception farklı siteler ve phytochromes yerelleştirilmiş havuzları moleküler mekanizmaları üzerinde kesin raporlar mekansal özel Fitokrom yanıtları sınırlı arabuluculuk yapmasını. Fitokrom photoperception belirli siteleri doku ve organa özgü yönleri fotomorfojenez ve lokalize Fitokrom havuzları, hücre-hücre sinyalizasyon downstream hedef genlerin farklı alt kümelerini meşgul düzenleyen bu hipotezler dayalı deneyler tasarlanmıştır. Biz seçici bir organ ya da doku-spesifik bir şekilde transgenik bitkiler içinde fonksiyonel phytochromes azaltmak için biyokimyasal bir yaklaşım geliştirdi. Çalışmalarımız transkripsiyonel aktivatör GAL4 Upstream Etkinleştirme Sırası (UAS) elemanının kontrolü altında bir gen ekspresyonu transaktivasyonunun sonuçları bir iki taraflı arttırıcı-trap yaklaşım dayanmaktadır<sup&gt; 3</sup&gt;. Biliverdin redüktaz (<em&gt; BVR</em&gt; UAS kontrolü altında) gen sessizce GAL4 transaktivasyonunun UAS-BVR ebeveynin yokluğunda korunur<sup&gt; 4</sup&gt;. UAS-BVR transgenik hattı ve belirli ifade GAL4-GFP arttırıcı tuzak hattı sonucu arasındaki genetik haçlar<em&gt; BVR</em&gt; Ile işaretlenmiş hücreleri gen<em&gt; GFP</em&gt; Ifadesi<sup&gt; 4</sup&gt;. BVR birikimi Fitokrom kromofor eksikliği Arabidopsis bitkiler sonuçları<em&gt; Planta</em<sup&gt; 5-7</sup&gt;. Bu nedenle, biz, sergi GAL4 bağımlı aktivasyon ürettiklerini transgenik bitkiler<em&gt; BVR</em&gt; Fitokrom biyokimyasal inaktivasyonu sonucu gen, yanı sıra GAL4 bağımlı<em&gt; GFP</em&gt; Ifadesi. Fotobiyolojik ve moleküler genetik analizler<em&gt; BVR</em&gt; Transgenik çizgileri, doku ve photoperception ilgili siteler ile ilgili organa özgü Fitokrom aracılı yanıtları içgörü verimli<sup&gt; 4, 7, 8</sup&gt;. Floresan Aktive GFP-pozitif Hücre Ayırma (FACS) arttırıcı-trap indüklenen<em&gt; BVR</emMikroarray analizi yoluyla hücre türüne özgü gen ekspresyon profili ile birleştiğinde&gt; ifade bitki protoplast mekansal özel Fitokrom yanıtları arabuluculuk dahil varsayılan downstream hedef genleri tanımlamak için kullanılmaktadır. Bu araştırma, ışık algı siteleri anlayışımız genişlemekte, çeşitli doku veya organların hafif düzenlenmiş bitki büyüme ve gelişme işbirliği ve karmaşık Fitokrom aracılı moleküler diseksiyon hücre-hücre sinyal kaskadlar ilerleyen üzerinden mekanizmaları.

Watch this Scientific Journal Video about Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana at JoVE.com

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

Mekansal özel Fitokrom yanıtlar moleküler temeli, doku ve organ spesifik Fitokrom eksiklikleri gösteren transgenik bitkiler kullanılarak araştırılmaktadır. Belirli hücrelerin izolasyonu mikroarray analizleri takip Sıralama mekansal özel Fitokrom yanıtları yer genleri tanımlamak için kullanılmaktadır Floresan-Aktif Hücre tarafından uyarılan Fitokrom kromofor tükenmesi sergiliyor.

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 ...

investigating-tissue-organ-specific-phytochrome-responses-using-facs

Nanyang Technological University

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JoVE Journal

Biology

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

15.7K Görüntüleme.  Michigan State University (MSU). Mekansal özel Fitokrom yanıtlar moleküler temeli, doku ve organ spesifik Fitokrom eksiklikleri gösteren transgenik bitkiler kullanılarak araştırılmaktadır. Belirli hücrelerin izolasyonu mikroarray analizleri takip Sıralama mekansal özel Fitokrom yanıtları yer genleri tanımlamak için kullanılmaktadır Floresan-Aktif Hücre tarafından uyarılan Fitokrom kromofor tükenmesi sergiliyor.

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

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

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

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

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

Floral-daldırma Dönüşüm Arabidopsis thaliana Inceleyin PTSO2: β-glucuronidase Reporter Gen İfadesi

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

Biyoloji

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

Hücre Tipi özel Yerelleştirme Analizi için Heterokaryon Tekniği

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

İnce Tabaka Kromatografisi (TLC) tarafından Arabidopsis thaliana Polar Glycerolipid Profil Gaz-Sıvı Kromatografi (GLC) ile birleştiğinde

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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.

Hücre Özgül Analizi<em&gt; Arabidopsis</em&gt; Floresans Aktif Hücre Ayırma kullanma Leaves

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

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

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

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

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

Erken evre Embriyolar verimli ve hızlı İzolasyon<em&gt; Arabidopsis thaliana</em&gt; Tohumlar

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

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 ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

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

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

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

Farklı ışık nitelikleri Arabidopsis thaliana büyüme davranış analizi

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

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Hücre türüne özgü Epigenome profil oluşturma

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Fare Karaciğerinde Hücre Tipine Özgü Gen Ekspresyonu Profiloluşturma

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...