GUS GUS-kusursuz bir protokol, Paja Sijacic ve Courtney Hollender paylaşımı için yararlı yorum ve Şekil 1, Detlef Weigel fotoğraflar için: Biz Chunxin Wang pTSO2 inşa için teşekkür ederiz . Z. L s laboratuvarda Araştırma, ABD Ulusal Bilim Vakfı (IOB0616096 ve MCB0744752) tarafından desteklenmektedir. ZL, Maryland Zirai Araştırma İstasyonu Üniversitesi tarafından kısmen destekleniyor.

I. Çiçekli daldırma Arabidopsis thaliana dönüşümü <ol><li> Yaklaşık 1 ay önce dönüşüm, pot başına yaklaşık 10-15 bitkiler ile 4-8 tencere (5 inç kare tencere) üzerine Arabidopsis tohumları ekmek. Plazmid inşa başına 4 tencere bitkilerin normal doğurganlık ile bitkiler için yeterli olacaktır. Doğurganlık azalmış mutant Arabidopsis dönüştürmek için varsa, buna göre kapların sayısını artırmak. </li><li> Bitkiler standart koşullar altında (22, 16 saat ışık ve 8 saat karanlık ° C) yetiştirilmektedir. Kısa gün veya daha düşük sıcaklık altında yetiştirilen bitkiler, bu dönüşüm için hazır hale çiçek ve daha uzun sürecektir. İyi bitki bakımı, başarılı bir dönüşüm için gerekli olan sağlıklı bitkiler, sağlayacaktır. </li><li> Bitkiler sadece cıvatalı ve çiçek başladı. Onlar şimdi, dönüşüm için hazır. Dönüşüm verimliliği artırmak için, dönüşüm 3-4 gün önce, en kısa sürede daha ikincil ateş oluşumunu teşvik etmek cıvatalı gibi ana gelişme sürgünler keserek. Bitkilerin dönüşüm önceki gün Su. Bu toprak, çiçek daldırma sırasında çok infiltrasyon medya emmek olacağını garanti eder. </li><li> : (Agrobacterium tumefaciens gerginlik GV3101 pTSO2 taşıma ile GUS, 50 mcg / ml Kanamisin: pTSO2 durumunda): İki gün önce dönüşüm, uygun antibiyotik içeren 40ml LB aşılamak: GUS inşa. PTSO2: GUS Kanamisin 3 direnç bahşeder NPTII gen sahip pBIN20 vektör klonlanmış. 28-30 ° C&#39;de sallayarak inkübatör gecede büyütün </li><li> Ertesi gün, 50 mg / ml Kanamisin içeren LB 400ml gecelik kültür transferi 8 ml. 28-30 ° C&#39;de sallayarak inkübatör gecede büyütün </li><li> Dönüşüm gününde, OD 600 Agrobacterium kültürünün yaklaşık 0.8 (0.5 1&#39;e arasında OD 600 kabul edilir) ulaştığında, 5000rpm 8 dakika Agrobacterium gecede kültürü aşağı doğru döndürün. </li><li> 1 L infiltrasyon medya Agrobacterium pelet yeniden süspanse (litre) (10mM MgCl 2,% 5 sakkaroz, 0.44mM 6-benzyladenine (BA),% 0.3 silwet L-77, 1x Gamborg vitamin çözüm ve otoklava su). Orta otoklava gereken değildir ama taze yapılmış olması gerekir. 1 L Agrobacterium içeren infiltrasyon orta bir yemek (8 &quot;x15&quot; x2 &quot;Pyrex cam tabak) içine dökün. </li><li> Pot haricindekileri (bitkiler kapalı düşme olmaz) ve bitkilerin tüm hava infiltrasyon çözüm içine daldırma, bir seferde bir tencerede 5 dakika tutun. Bitkinin tüm hava infiltrasyon orta dalmış iken, toprak, toprak üzerinde mantar büyüme şansını azaltmak için herhangi bir infiltrasyon medya içinize izin kaçının. </li><li> Daldırma sonra, bir tepsi kağıt havluyu birkaç kat üzerinde kendi tarafında tüm daldırma tencere yerleştirin. Kağıt havlu infiltrasyon medya erişim miktarı içinize çekin. Tepsi yüksek nem sağlamak için naylon ile örtün. Bitki büyüme odasına tepsiye yerleştirin. </li><li> Ertesi gün, dik plastik wrap ve kağıt havlu, yer ve tencere çıkarın. 4-5 gün daha bu bitkiler su etmeyin. Daha sonra, tohum kadar normalde bitkilerin bakımı ve toplu olarak tohumları toplamak. </li><li> 4 gram serin agar, otoklav ve 1M NaOH ile 5,8, 500 ml su, pH içinde çözülür, MS 50 mg / ml Kanamisin (ağırlık 2.2G Murashige içeren orta ve bazal tuz vitamini olmadan Skoog levhalar (150x15mm petri) dökün ) 50 mg / ml nihai konsantrasyonu Kanamisin ekleyin. Tabaklar gerekli ° C ye kadar 4 tutulur. </li><li> Antibiyotiğe dayanıklı transgenik bitkiler için seçmek için en az 20.000 tohum ekrana ihtiyacı var. 0.1 gram yaklaşık 5000 tohum. Ağırlık ve tohum miktarı tahmin. </li><li> 15 ml Falcon tüp 30 saniye boyunca% 70 etanol ile yıkayın tohum sterilize edin. Tohumları bir kez steril su ile yıkayın. 01:10 seyreltme mağaza satın aldı,% 5 çamaşır suyu (Clorox) içeren bir çözüm için tohum 30 saniye bekletin. Tohumları 3-6 kez steril su ile durulayın. Her MS (Kanamisin) plaka üzerine pipet yaklaşık 5000 tohumlar, steril kaput, Micropore 3M bant ile kirlenmesini önlemek amacıyla plakaları, plaka üzerinde eşit tohum yayıldı. </li><li> 4 plakaları inkübe ° C, 3-4 gün boyunca karanlıkta, sonra bir odaya (aynı bitki büyüme odası olabilir) plakaları aktarmak. Yaklaşık 14 gün sonra, transgenik fide yeşil kalır ama transgenik olmayan soluk dönüm ve ölüyor. Yavaşlama orta kökleri dışarı çekerek ve toprağın yerleştirdiler transgenik fideler toprağa aktarın. </li></ol> II. PTSO2 incelenmesi: GUS ekspresyonu <ol><li> Hasat pTSO2: GUS transgenik fide ve% 90 aseton, bir bardak sintilasyon flakon veya mikrofuge&#39;de tüpleri soğuk koyunbuz üzerinde tutulur. Biri büyük bir sayıda numune analiz eğer Polistiren microtiter plakalar (polipropilen) de kullanılabilir. </li><li> Tüm örnekler hasat sonra, 20 dakika boyunca oda sıcaklığında şişeleri. Bu süre zarfında x-Gluc (% 0.2 Triton X-100, 50mm NaHPO 4 Tampon pH7.2, 2mm Potasyum Ferrocyanide, 2mm Potasyum ferrisiyanür) ve buz üzerinde yer olmadan taze boyama tampon oluşturur. </li><li> Örneklerden aseton çıkarın ve boyama tamponu ekleyin. </li><li> X-Gluc boyama solüsyonu (% 0.2 Triton X-100, 50mm NaHPO 4 Tampon pH7.2, 2mm Potasyum Ferrocyanide, 2mm potasyum ferrisiyanür, 2mm x-gluc) olun. Örneklerinden boyama tamponu çıkarın ve x-Gluc örnekleri içeren boyama tamponu ekleyin. </li><li> 15 ila 20 dakika arasında bir vakum altında örnekleri Infiltrate. Vakum yavaşça serbest bırakın ve tüm örnekleri boyama çözüm yüzeyin altında lavabo doğrulamak. Gerekirse vakum yayımlandıktan sonra, tüm örneklerde lavaboya kadar infiltrasyon tekrarlayın. </li><li> 37 inkübe örnekleri ° C x-Gluc boyama tampon geceleme. </li><li> Inkübatör örnekler çıkarın ve boyama tamponu çıkarın. Her değişiklik için 30 dakika oda sıcaklığında (% 20,% 35 ve% 50 etanol), ardışık etanol serisi örnekleri yıkayın. </li><li> Oda sıcaklığında 30 dakika içinde FAA fiksatif (% 50 Etanol,% 5 Formaldehit,% 10 asetik asit, geri kalanı su) dokuların sabitleyin. FAA çıkarın ve% 70 etanol ekleyin. Bu noktada dokuların görüntülendi olabilir ve bir dijital kamera ile donatılmış bir diseksiyon mikroskobu altında fotoğraflandı. TSO2 (Şekil 1) ifade edilir, koyu mavi renk gösterir . Alternatif olarak, dokuların, 4 ° C daha sonra izlenmek üzere saklanabilir. <img src="/files/ftp_upload/1952/1952fig1.jpg" alt="Şekil 1" /> Şekil 1 pTSO2:: GUS transgenik fidan ifade (A), yan kök (B), ve kök ucu (C). TSO2 organizatörü, genç ve bölme dokularda son derece aktif olduğunu unutmayın . (C) gösterilen sporadik ifade deseni, belirli bir hücre döngüsü evreleri olarak ifade genlerin tipik bir örneğidir. </li></ol> Temsilcisi Sonuçlar Doğru bir şekilde yapılırsa, dönüşüm verimliliği yaklaşık% 0.1-0.2 olmalıdır. Başka bir kelime, bir, 5000 tohum eleme 5-10 transgenik fide almalısınız. Ortalama, yaklaşık 100 transgenik çizgileri vahşi tip bitkiler 4 tencere dönüştürerek elde edilebilir. Için pTSO2 içeren transgenik fide: GUS inşa 3, GUS faaliyet yansıtan koyu mavi renk, genç yaprak, sürgün apeks, kök ucu ve lateral kök primordia (Şekil 1) de dahil olmak üzere aktif olarak bölünen hücreleri bulunur . Üniform olmayan sporadik desen hücre döngüsü faz-spesifik ifade karakteristik.

<ol>
	<li>Clough, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+agrobacterium-mediated+germ+line+transformation.">Floral dip: agrobacterium-mediated germ line transformation.</a> Methods Mol Biol. 286, 91-91 (2005).</li><li>Clough, S. J., Bent, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Floral+dip:+a+simplified+method+for+Agrobacterium-mediated+transformation+of+Arabidopsis+thaliana.">Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.</a> Plant J. 16 (6), 735-735 (1998).</li><li>Wang, C., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+ribonucleotide+reductases+are+critical+for+cell+cycle+progression,+DNA+damage+repair,+and+plant+development.">Arabidopsis ribonucleotide reductases are critical for cell cycle progression, DNA damage repair, and plant development.</a> Plant Cell. 18 (2), 350-350 (2006).</li></ol>

Dönüşüm verimliliği aşağıda tartışılmıştır çok farklı faktörler tarafından belirlenir: <ol><li> Birincil öneme sahip bitkilerin sağlık ve yaşlarına. Üreten çok sayıda olgunlaşmamış çiçek tomurcukları yaklaşık bir aylık bitkiler idealdir. Dönüşüm ikincil ateş oluşumu 3-4 gün önce teşvik etmek birincil sürgünler kapalı Kırpma dönüşüm verimliliği artacaktır. Önceki bitkiler transformants doğuran, ancak daha düşük bir oranda. </li><li> Bitkilerin Doğurganlık ?...

<table><tbody><tr><td>6-benzyladenine</td><td>Sigma-Aldrich</td><td>852430</td><td></td></tr><tr><td>Silwet-77</td><td>Crompton Corp.</td><td></td><td>Toxic, wear gloves</td></tr><tr><td>Murashige and Skoog Basal medium (MS)</td><td>Sigma-Aldrich</td><td>M5519</td><td></td></tr><tr><td>Gamborg&amp;rsquo;s vitamin solution 1000X</td><td>Sigma-Aldrich</td><td>G1019</td><td></td></tr><tr><td>X-Gluc (5-bromo-4-chloro-3-indolyl &amp;#946;-D-glucuronide cyclohexamine salt</td><td>Biosynth International, Inc</td><td>B-7300</td><td>Toxic, light sensitive keep in the dark</td></tr><tr><td>Micropore 3M Tape</td><td>Fisher Scientific</td><td>19027761</td><td></td></tr></tbody></table>

floral-dip transformation of arabidopsis thaliana to examine ptso2::&beta;-glucuronidase reporter gene expression

Bir organizmanın içine yabancı genlerin tanıtmak için yetenek, modern biyoloji ve biyoteknolojinin temelini oluşturur. Model çiçekli bitki<em&gt; Arabidopsis thaliana</em&gt; Çiçek daldırma dönüşüm yöntemi<sup&gt; 1-2</sup&gt; Basitlik, verimlilik ve düşük maliyet nedeniyle önceki tüm yöntemler yerini almıştır. Özellikle genç çiçeklenme, sürgünler<em&gt; Arabidopsis</em&gt; Bitkilerin bir çözüm batırılmış<em&gt; Agrobacterium tumefaciens</em&gt; Belirli plazmid yapıları taşımak. Daldırma sonra, bitkilerin normal büyüme ve verim tohumları küçük bir yüzdesi, yabancı gen ile dönüştürülmüş ve antibiyotik içeren orta için seçilebilir iade edilir. Önemli ölçüde kolaylaştırdı Bu çiçek daldırma yöntemi<em&gt; Arabidopsis</em&gt; Araştırma ve bitki gen fonksiyon anlayışımıza büyük katkıda bulunmuştur. Bu çalışmada, biz, bir muhabir gen dönüştürmek için çiçek daldırma yöntemi kullanın<em&gt; Β-glucuronidase (GUS)</em&gt; Kontrolü altında<em&gt; TSO2</em&gt; Organizatörü.<em&gt; TSO2</em&gt; Ribonukleotid redüktaz (RNR) küçük altbirim için kodlama<sup&gt; 3</sup&gt;, Hücre döngüsü, hücre döngüsünün S-fazında dNDP biyosentezi için gerekli bir düzenlenir gen. İncelenmesi<em&gt; GUS</emTransgenik&gt; ifadesi<em&gt; Arabidopsis</em&gt; Fide olduğunu gösteriyor<em&gt; TSO2</em&gt; Aktif dokularda bölünerek ifade edilir. , Yüksek okul ve üniversite düzeylerinde araştırma için değil, aynı zamanda eğitim için değil, sadece bildirilen deneysel yöntem ve malzemeleri kolaylıkla adapte edilebilir.

Watch this Scientific Journal Video about Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::Î²-glucuronidase Reporter Gene Expression at JoVE.com

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

Bu makale, çiçek-daldırma yöntemi göstermektedir.<em&gt; Agrobacterium tumefaciens</em&gt; Aracılı dönüşüm<em&gt; Arabidopsis thaliana</em&gt;. Bir hücre döngüsü düzenlenir organizatörü muhabiri tanıtarak,<em&gt; PTSO2: β-glucuronidase (GUS)</em&gt; Içine<em&gt; Arabidopsis</em&gt;, Bir transgenik fide GUS muhabir ifade nasıl algıladığı göstermektedir.

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 ...

floral-dip-transformation-arabidopsis-thaliana-to-examine-ptso2

Research

JoVE Journal

Biology

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

36.1K Görüntüleme.  University of Maryland College Park. Bu makale, çiçek-daldırma yöntemi göstermektedir. Agrobacterium tumefaciens Aracılı dönüşüm Arabidopsis thaliana. Bir hücre döngüsü düzenlenir organizatörü muhabiri tanıtarak, PTSO2: β-glucuronidase (GUS) Içine Arabidopsis, Bir transgenik fide GUS muhabir ifade nasıl algıladığı göstermektedir.

Video: Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Gibson Meclisi ve Gen Tabancası kullanarak Tütün geçici Gen İfadesi

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Çevre

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the transgene, as multi-copy integrations are often subjected to gene silencing. The Gateway-compatible binary vector based on bacterial artificial chromosomes (pBIBAC-GW), like other pBIBAC derivatives, allows the insertion of single-copy transgenes with high efficiency. As an improvement to the original pBIBAC, a Gateway cassette has been cloned into pBIBAC-GW, so that the sequences of interest can now be easily incorporated into the vector transfer DNA (T-DNA) by Gateway cloning. Commonly, the transformation with pBIBAC-GW results in an efficiency of 0.2&#8211;0.5%, whereby half of the transgenics carry an intact single-copy integration of the T-DNA. The pBIBAC-GW vectors are available with resistance to Glufosinate-ammonium or DsRed fluorescence in seed coats for selection in plants, and with resistance to kanamycin as a selection in bacteria. Here, a series of protocols is presented that guide the reader through the process of generating transgenic plants using pBIBAC-GW: starting from recombining the sequences of interest into the pBIBAC-GW vector of choice, to plant transformation with Agrobacterium, selection of the transgenics, and testing the plants for intactness and copy number of the inserts using DNA blotting. Attention is given to designing a DNA blotting strategy to recognize single- and multi-copy integrations at single and multiple loci.

Using a pBIBAC-GW binary vector makes generating transgenic plants with intact single-copy insertions, an easy process. Here, a series of protocols is presented that guide the reader through the process of generating transgenic Arabidopsis plants, and testing the plants for intactness and copy number of the inserts.

Transgenik bitkiler BIBAC-GW ikili vektör kullanarak tek kopya ekleme ile oluşturma

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

Genetik

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

İndüklenebilir, hücre türüne özgü ifade Arabidopsis thaliana aracılığıyla LhGR-aracılı Trans-harekete geçirmek

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

Bitkilerde etkin bir şekilde birden fazla Chimeric floresan Fusion proteinlerin ortak ifade

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Kimya

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 ...

Biyoloji

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

Proteinlerin içinde Planta serilerinde Plasmodesmal yerelleştirme tanımlaması

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

İmmünoloji ve Enfeksiyon

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 ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Keten Çiçekli-Dip Dönüşüm (<em&gt; Linum usitatissimum</em&gt;) Yüksek Dönüşüm Oranı ile Transgenik progeny&#39;lerinde oluşturmak için

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Bir Lüminesans Saat Reporter ile Arabidopsis Protoplasts transfekte içinde Sirkadiyen fenotipleri Hızlı Analizi

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

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

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

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

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

Arabidopsis montaj için çok yönlü bir yöntem Intravital zaman hata görüntüleme için yaprakları

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

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

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