Bu çalışma, ABD Ulusal Bilim Vakfı Christoph Benning bir hibe ile desteklenmektedir.

1. Lipid Ekstraksiyon <ol><li> 30 mg agar üzerinde yetiştirilen bitkiler 4 hafta eski Arabidopsis yaprakları orta ya da toprak katılaşmış ve 1,5 mL polipropilen reaksiyon tüpleri içine transfer hasat tarafından lipid ekstraksiyon başlayın. Taze yaprakları flaş sıvı nitrojen içinde dondurulmuş ve -80 ° C&#39;de saklanabilir. </li><li> Metanol oluşan solvent 300 mcL çıkarma, kloroform ve formik asit (20:10:01, v / v / v) her numune ekleyin. (Bir boya Shaker ya da benzeri kullanarak) 5 dakika çalkalanır. </li><li> 0.2 M fosforik asit (H 3 PO 4), 1 M potasyum klorür (KCl) ve vorteks kısaca 150 mcL ekleyin. </li><li> 1 dakika boyunca oda sıcaklığında 13.000 xg&#39;de santrifüjleyin. Alt kloroform fazında çözünmüş Lipidler TLC plakaları damlatılan olacaktır. </li></ol> 2. İnce Tabaka Kromatografisi (TLC) 15 <ol><li> Batığın 0.15 M amonyum sülfat ((NH 4) 2 SO 4) solüsyonu içine 30 saniye için şerit yükleme 20cmX20cm ile silika jel kaplı TLC plağı, TLC plakaları hazırlamak için, 30 saniye boyunca daldırarak sonra, en az 2 gün boyunca tabak kuru kapalı bir kapta. Etkinleştirme sırasında diğer glycerolipids ayrılması için gerekli phosphatidylglycerol protonates sülfürik asit, amonyum süblimasyon arkasında bırakır. </li><li> Deney günü etkinleştirmek 120 fırında pişirme tarafından plakaları TLC ° C, 2,5 saat süreyle. </li><li> Oda sıcaklığına kadar aktive edilmiş plakalar Soğuduktan sonra, plaka üzerinde kromatogram kökeni (plaka kenarından 1,5 cm) düz bir çizgi çizmek için bir kalem kullanın. </li><li> Davlumbaz, yavaş yavaş bir akışı altında N 2 200 mcL sarı plastik ipuçları ile 20 mcL pipet kullanarak alt kloroform faz 3 X 20 mcL lipid özü teslim. Bu amaçla, bir Tygon Boru N 2 tank, regülatör bağlıdır. Spot çapı 1 cm&#39;den daha küçük tutunuz. Her plaka 10 örnek (sonraki GLC analiz planlanan olduğunda) kadar tutabilir. </li><li> Lipid davlumbaz tamamen kuru noktalar gibi, aseton, toluen, su (30 ml: 7.5 ml 91 ml) oluşan gelişmekte olan solvent hazırlar. Ortam bağıl nem yüksek ise, ayrılık etkilenebilir. Bu durumda istenen ayırım elde (7.0 ml: 30 ml 91 ml) su vermek azaltılmalıdır. </li><li> 80 ml çözücü gelişmekte olan ve aşağı bakacak şekilde örnek sonuna tankına plaka odası (H: W = 27.0: 26.5 7.0 cm / cm / cm L) gelişmekte olan bir yapışmalı, TLC içine dökün. Tank kelepçe kullanarak Seal. Solvent plaka yükselmek ve lipidler ayrılacaktır. Geliştirme zamanını, oda sıcaklığında yaklaşık 50 dakika mesafededir. </li><li> Çözücü ön plaka üstten 1 cm ulaştığında, dikkatlice plaka tanktan çıkarın ve yaklaşık 10 dakika için davlumbaz tamamen kuru. </li><li> TLC ayrılmış Lipidler kantitatif analiz ya da sülfürik asit veya α-naftol geri dönüşümsüz lekeli iyot ile ya da geri dönüşümlü kısaca lekeli olabilir. <ol><li> Sülfürik asit yanmaları:% 50 sülfürik asit, davlumbaz ve fırında 120 ° C&#39;de 15 dakika (Şekil 2A) spreyli cam bir şişede su spreyi plaka. </li><li> glikolipitler için α-naftol boyama:% 2.4 (w / v)% 10 α-naftol (v / v), sülfürik asit,% 80 (v / v) etanol ve fırında 120 ° C plaka uygulaması için 3-5 glikolipid bantları kadar dakika (Şekil 2B), pembe veya mor lekeli. Aşırıtedavi reaktif sülfürik asit varlığı nedeniyle tüm lipidler yanmaları için yol açacaktır. </li><li> İyot boyaması: davlumbaz plaka, iyot kristalleri ile kapalı bir TLC (lipidler görünür olana kadar iyot buharı ile atmosferin doygunluk önde gelen altındaki bir tepsi) tankı içine yerleştirin. Iyot iyot kovalent çoklu doymamış yağ asitleri (Şekil 2C) değiştirebilir gibi çok uzun plaka maruz bırakmayın. Alternatif olarak, örnek şeritli serpiştirilmiş sadece standart şerit N 2 ayrı standart şerit üzerinde üflenir iyot kristalleri ile Pasteur pipeti takılı bir cam yünü kullanılarak boyandı olmalıdır, lipidlerin herhangi bir oksidasyon önlemek için. </li></ol></li></ol> 3. Yağ Asil metilester (FAME) Reaksiyon 16 <ol><li> Bir jilet ile TLC plağı silika çevreleyen lipit noktalar tespit çıkarın. Lipit içeren silika kazıyın ve Teflon (PTFE) kaplı vidalı kapaklı cam tüp içine bir huni kullanarak silis tozu aktarımı. </li><li> Cam pipet ile her bir örnek susuz metanol 1 ml 1 N hidroklorik asit (HCl) ekleyin. </li><li> 200 mcL sarı plastik ucu ile 200 mcL pipet kullanarak iç standart olarak her numune için 200 mcL pipet kullanarak 100 mcL 50 mg mL -1 pentadecanoic asit (15:00) ekleyin. Bir tüp kontrol olarak sadece pentadecenoic metanol HCl asit ile tutun. Cam tüpler Teflon kaplı kapakları sıkıca kapatın. </li><li> Inkübe gl25 dakika boyunca 80 ° C su banyosu göt tüpleri. Solvent buharlaşma olmaz böylece Tüpler mühürlü olması gerekir. </li><li> Tüpler soğutulduktan sonra, şiddetle 1 ml hekzan ve vorteks 1 ml% 0.9 sodyum klorür ekleyin. 3 dakika için 1000xg numune santrifüjleyin. </li><li> Davlumbaz, Pasteur pipeti ile örnek hekzan / üst tabakayı kaldırmak ve yeni bir 13x100 mm cam tüp içine yerleştirin. </li><li> N 2 yavaş bir akışı tamamen kuruması beklenmeden altında hekzan buharlaşır. </li><li> 60 mcL hekzan çıkan yağ açil methylesters s eritin. Örnekleri otosampler şişeleri ve kapağı sıkıca içine aktarın. Numuneler, kısa vadeli ve -20 ° C ° C bir kaç gün için 4 saklanabilir. </li></ol> 4. Gaz-Sıvı Kromatografi (GLC) 10 <ol><li> GLC başlamadan önce, helyum, hidrojen ve hava tüpleri dolu olduğundan emin olun. </li><li> Yeterli hekzan, solvent rezervuar eklendi olmalı ve atık kabını boş olmalıdır. Yağ açil methylesters ayrılması için, makineyi bir DB-23 sütun ekleyebilirsiniz. </li><li> Otosampler yerleştirmek gözlerdir. Sistemi bilgisayar GLC ChemStation yazılımını başlatın. </li><li> Giriş sıcaklığı 250 ° C helyum akış hızı ve 48.6 mL dak -1 21.93 psi basınç. Ayarlayın Split oranı 30.0: 1. </li><li> Fırın sıcaklığı 140 ° C &#39;de 2 dakika süreyle başlangıçta ortaya konan ve 160 ° C - 25 ° C min -1 hızında kaldırdı . Ardından 160 artış sıcaklığını ayarlamak ° C ila 250 ° C hızında 8 ° C min -1 ve 250 tutun ° C - 140 azalma takip 4 dk ° C hızında 38 ° C min - 1. Bir vadede yaklaşık 21 dakika sürer. </li><li> Alev iyonizasyon dedektör sıcaklığı 270 ° C ve hidrojen debisi 30.0 mL dak -1, hava debisi 400 mL dak -1 ve helyum debisi 30.0 mL dak -1. </li><li> Şişeleri ve çalışma sıra tabloda örnek isimleri numarasını girin. 10 mcL enjektör, flakon başına 2 mcL örnek enjekte etmek için ayarlayın. </li><li> Cihaz hazır olduğunda, çalışma dizisi başlatmak. </li></ol> 5. Temsilcisi Sonuçlar: 4 haftalık Arabidopsis fide TLC ayrılmış lipitlerin geri dönüşü olmayan boyama örnekleri Şekil 2&#39;de gösterilmiştir. Sülfürik asit lekeli lipidler (Şekil 2A) kömürleşmiş ve kahverengi lekeler olarak görünür. α-naftol diğer polar lipid (Şekil 2B) sarı leke α-naftol pembe-mor bir renk taşıyan boyanmış vb Glikolipitler SQDG DGDG gibi MGDG glikolipitler leke tercih edilir. Iyot boyama geri dönüşümlüdür ve lipidler, iyot buharlaşır (Şekil 2C) gibi kısa bir zaman içinde kaybolur sarımsı bir renk verir. Lekesiz lipidler Lipidlerin yıkmak azaltmak için tercih edilir rağmen Kısaca iyot lekeli lipidler GLC analiz tabi tutulabilir. Başarılı bir şekilde yapılırsa, farklı Yağ açil metilester temsil eden ayırt edici sinyaller (Şekil 3) GLC sonra gözlenen olacaktır. Yağ açil metilester kısa karbon zinciri ve daha az çift bağ ile DB-23 sütun kullanarak kısa tutma süresi var. Yağ açil metilester profil değişmiş lipid kompozisyonu ile mutantlar tanımlamak için hassas bir araçtır. Şekil 4&#39;te, vahşi tip 18 ile karşılaştırıldığında tgd4-1 mutant MGDG18: 3 yağ asidi molar oranda azalmıştır. Yağ açil metilester bir lipid sınıf için tüm lipid sınıflarının mol mol bölerek, her lipid molar oranı hesaplanır. Örneğin, MGDG molar oranını hesaplamak için: (MGDG) mol% = Σ [Fames (MGDG)] / Σ [Fames (toplam)] x100% Yabani tip ve mutant her lipid sınıfı molar oranları mukayese edilebilir. Örneğin, tgd4-1 mutant MGDG ve PG göreceli miktarları artmış ama DGDG ve PE (Şekil 5) 18 miktarda azalma var. <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="Şekil 1" /> Şekil 1 Arabidopsis fide kullanarak polar lipit analizi akış şeması. Toplam lipidler, 4 haftalık Arabidopsis fide çıkarılan ve TLC ayrılır . Ayrılmış lipidler GLC analizi ile takip transesterifikasyon TLC plağı kazınmış olabilir. <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="Şekil 2" /> Şekil 2 TLC plakaları üzerinde lipitlerin ayrılması. 35 mg lipid özleri (taze ağırlık) vahşi tip fide TLC ayrılmış ve sülfürik asit (A), α-naftol (B) veya iyot buharı (C) ile boyanan. Üç tekrarlar, her boyama yöntemi gösterilmiştir. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, fosfatidilkolin, PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, fosfatidilinositol; SQDG, sulfoquinovosyldiacylglycerol. <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="Şekil 3" /> Şekil 3 Yağ Asitleri Methylesters GLC analizi (Fames) vahşi tip MGDG türetilmiştir. Fames 30 m kılcal sütun üzerinde ayrılır ve alev iyonizasyon tarafından tespit edilir. Pentadecanoic asit (15:00) bir iç standart olarak kullanılır. <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="Şekil 4" /> Şekil 4 vahşi tip Süt2 (beyaz sütunlar) ve mutant tgd4-1 (siyah sütunlar) MGDG Yağ asidi profili. Yağ asitleri çift bağların sayısına göre takip karbonlar sayısı olarak sunulmaktadır. Üç tekrarlar ortalama ve standart sapmaları gösterilmiştir. <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="Şekil 5" /> Şekil 5 vahşi tip Süt2 (beyaz sütunlar) Polar lipidler kompozisyon ve tgd4-1 mutant (siyah sütunlar). Üç tekrarlar ortalama ve standart sapmalar hata bar gösterilmiştir.

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TLC GLC ile birleştiğinde tesislerinde polar lipid kantitatif analiz için sağlam ve hızlı bir araç sağlar. Lipid bileşimi küçük değişiklikler tespit edilebilir, bu nedenle bu yöntem, polar lipid metabolik yollar 1,20 engelli mutantlar büyük ölçekli tarama için kullanılır olmuştur . Bu yöntem ayrıca, substrat olarak polar lipidler kullanan enzimler izleme faaliyetleri için yaygın olarak kullanılır . 2,6,7 Yaprakları yanı sıra, bu kök ve tohumları veya kloroplast ve mitokondri gibi hücre içi fraksiyonları gibi diğer bitki dokularının lipid kompozisyonu da aynı şekilde tespit edilebilir. Burada kullanılan çözücü sistemi (aseton, toluen, su), bitkilerde glikolipitler ve fosfolipidlerin ayrılması için optimize edilmiştir. Ancak, tgd1, 2,3,4 mutantlar ve izole kloroplastların tetragalactosyldiacylglycerol PC ile çalışırken TGDG PE ile birlikte çalışır. Bu durumda, kloroform, metanol, asetik asit ve su (85: 20: 10: 4, v / v / v / v) ile çözücü sistem 13 kullanılır . Bazen iki farklı çözücü sistemlerini kullanarak iki boyutlu TLC, daha fazla ayrı glikolipitler ve fosfolipitler 19 yapılır . Buna ek olarak, bitki dokularında doğrudan TLC 5 başlangıç ​​ayrımı olmadan toplam yağ asidi profili belirlemek için GLC takip FAME reaksiyon tabi olabilir. Gösterdi TLC GLC sisteminin yanı sıra, lipid profili kullanılan başka bir yöntem, doğrudan elektrosprey iyonizasyon tandem kütle spektrometresi 17 dayanmaktadır . Bu yöntemde ilk kromatografik ayırma özü lipitlerin atlanır. Ancak, bu yöntem, laboratuvar veya mutant taraması için rutin analizler için daha az kullanışlı hale getirir pahalı ekipman ve deneyimli personel gerektirir.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

Biyolojik membranlar ayrı hücreler çevreden. Fosfatidilkolin veya phosphatidylethanolamine, hücreleri ve çevreleri arasındaki kimyasal değişimi için hem sınırları ve arayüzleri olarak hareket form bilayeri membranlar gibi çok hücreli bitki ve hayvanlar, glycerolipids, tek bir hücre. Hayvanların aksine, bitki hücreleri fotosentez için özel bir organel kloroplast var. (MGDG) monogalactosyldiacylglycerol digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) 4: kloroplastın karmaşık membran sistemi yani benzersiz glycerolipids, fosfor eksikliği glikolipitler içerir. Bu lipitlerin rolleri sadece yapısal dışındadır. Bu glikolipitler ve diğer glycerolipids fotosistem I ve II fotosentez 8,11 glycerolipids katılımı gösteren kristal yapıları bulundu. Fosfat açlık sırasında, DGDG fosfolipitler 9,12 kaybını telafi etmek extraplastidic membranlar aktarılır. Bu lipitlerin biyosentezi ve fonksiyon bilgimizin Arabidopsis thaliana 14 ile biyokimyasal ve genetik çalışmalar bir arada elde edilmiştir. Bu çalışmalar sırasında, polar lipid analizi için basit bir prosedür lipid mutantlar tarama ve analiz için gerekli ve detaylı olarak özetlenen olacaktır. Bir yaprak lipid özü ilk ince tabaka kromatografisi (TLC) ile ayrılmış ve glycerolipids iyot buharı ile geri dönüşümlü lekeli. Bireysel lipidler TLC plağı koparılmış ve alev iyonizasyon algılama (FID GLC) (Şekil 1) ile birleştiğinde gaz-sıvı kromatografisi ile analiz edilir yağ açil methylesters (Fames) dönüştürülür. Bu yöntem, mutant tarama için güvenilir bir araç olduğu kanıtlanmıştır. Örneğin tgd1, 2,3,4 endoplazmik retikulum-plastid lipid kaçakçılığı mutantlar anormal bir galactoglycerolipid birikimine dayalı bulunmuştur: trigalactosyldiacylglycerol (TGDG) ve göreli 18:03 miktarda bir azalma (karbonlar: double bağ) 3,13,18,20 zarı lipidler yağ açil grupları. Bu yöntem ayrıca substrat 6 olarak lipidler kullanarak proteinlerin enzimatik faaliyetleri belirlemek için uygulanabilir.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

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

Polar lipid özler bireysel glycerolipids Kompozisyon ve yağ asidi kompozisyonu basit ve sağlam bir lipid profil deneyde belirlenir. Bu amaçla, glycerolipids ince tabaka kromatografisi ile izole edilir ve onların açil grupları transmethylation tabi. Yağ açil methylesters gaz-sıvı kromatografisi ile sayısallaştırılmış.

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 ...

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Research

JoVE Journal

Biology

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

36.4K Görüntüleme.  Michigan State University. Polar lipid özler bireysel glycerolipids Kompozisyon ve yağ asidi kompozisyonu basit ve sağlam bir lipid profil deneyde belirlenir. Bu amaçla, glycerolipids ince tabaka kromatografisi ile izole edilir ve onların açil grupları transmethylation tabi. Yağ açil methylesters gaz-sıvı kromatografisi ile sayısallaştırılmış.

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

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

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 ...

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::&amp;#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 ...

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 ...

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 ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

Ince katman Kromatografi takip Escherichia coli (p) ppGpp çoklu In vivo ölçümleri Için mikrotiter bulaşık Radiolabeling kullanma

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

ERL1 Dinamiğinin Konfokal Görüntüleme ile Uzay-Zamansal Olarak İncelenmesi

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Yazar Spotlight: Stoma Soy Hücrelerinde Membran Kaçakçılığı Olaylarını İncelemek için Görüntü Tabanlı Yöntemler  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Stoma Soy Hücrelerinde Membran Kaçakçılığı Olaylarını İncelemek için Görüntü Tabanlı Yöntemler

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...