이 작품은 시작 그랜트 OKV에게 수여 코넬 대학 농업 실험 스테이션 (CUAES) NYC - 125433 해치 그랜트와 코넬에서 지원했습니다

1 부. 성장 식물 고체 매체의 준비. 우리는 일반적으로 집중 Murasige와 Skoog (MS) 매체에 문화 공장 1 % 자당 및 0.7 %의 한천과 함께 보충. 그것은 식물이 건강하게 유지하는 필요한 모든 재료가 포함되어 있습니다. MS의 1L, 미디어 산도 5.7 준비 <ol><li> 1.5 2.0L autoclavable 병에 물 800 ML에 MS 분말 2.15 g을 추가합니다. </li><li> 병에 감동 막대를 삽입하고 감동 접시에 매체를 교반하여 MS 분말을 풀다. </li><li> 교반하는 동안, 추가 드롭 들러, 1N 코는 5.7으로 MS 매체의 산도를 조정합니다. </li><li> 자당의 10g를 추가, 감동 유지. </li><li> 한천의 7g 추가 *. </li><li> 1000 ML에 준비 매체의 최종 볼륨을 조정하고 autoclaving하여 소독 .* </li><li> 장소 감동 접시에 매체를 autoclaved하고 교반하면서 그것을 진정 그 한천은 병의 바닥에 침전되지 않습니다. ~ 60 ° C. *로 매체를 쿨 </li><li> 100mm 페트리 접시에 붓고. 4 접시 보관 ° C. </li></ol> * 팁 1 : 한천을 분해하지 마십시오. 그것은 autoclaving 동안 solubilize 것입니다. * 팁 2 : autoclaving 동안 병에 교반 막대를 유지하는데, 나중에 필요합니다. * 팁 3 : 10 s에 대한 autoclaved 병에 손을 유지할 수있다면, 그것은 매체 충분히 식었 당신이 접시를 따르고 시작할 수있다는 것을 의미합니다. 2 부. 식물과 성장 조건. <ol><li> Arabidopsis thaliana의 씨앗을 소독 <ol><li> eppendorf의 microcentrifuge 튜브에 씨앗 100 MG를 삽입하고 70 % 에탄올 1 ML를 추가합니다. 잘 믹스 2 분 알을 품다. , 씨앗을 스핀 다운 뜨는을 대기음. </li><li> 1.8 %의 표백제 용액 1 ML 추가 * 0.1 % 트윈 - 20를 포함. 10 분 섞는다. 종자 및 대기음 표백 용액을 스핀. </li><li> 이 단계는 층류 후드에서 수행되어야한다. 씨앗을 멸균 물 1 ML 추가, 잘 섞는다. 물을 기음, 씨앗을 스핀. 이 단계에게 5 회 반복합니다. </li></ol></li><li> 1 단계 접시 (100 씨앗 / 플레이트)의 준비에 씨앗을 퍼뜨린다. </li><li> 4 24-48 H에 대한 씨앗을 품고 번호판을 계속 ° 충화 동안 짙은 어둠 속에서 C. </li><li> 19분의 23 ° C 빛 / 어둠의 온도 정권 8 H light/16 H 어두운 photoperiod (250 μmol m -2 S -1의 광합성 광자 선속 밀도에서)와 함께 성장 챔버에서 14 일 동안 식물을 성장하고 75 % 상대 습도. </li></ol> * 팁 1 : 블리치 솔루션 지어낸입니다 희석 가정 Clorox하여 6 % 나트륨 차아 염소 산염을 포함하고 0.1 %의 최종 농도 트윈 - 20를 추가. 부 3. Arabidopsis 모종의 protoplasts의 고립. <ol><li> 슬라이스 필터 - 살균 TVL 솔루션의 15 ML에 신선한 면도날로 14 일 된 모종의 2g. 우리는 일회용 멸균 배양 접시에 식물 자료를 들어온다. </li><li> 200 ML의 비커에 잘게 조직을 전송, 필터 살균 효소 솔루션, 소용돌이 효소 솔루션으로 조직 믹스가 parafilm 및 알루미늄 호일로 커버하도록 비커 20 ML를 추가합니다. </li><li> 16-18 H. 위해 상온에서 어두운 35 RPM에서 식물 조직 쉐이크 </li><li> 치즈 천 8 층, W5 용액에 미리 서부 유럽 표준시를 통해 거를 50 ML 팔콘 튜브에 공개 protoplasts를 수집합니다. </li><li> W5 솔루션의 15 ML과 천을 세탁하여 치즈 천으로 한 번 더에서 체의 protoplasts. </li><li> 조심스럽게 W5 솔루션의 10 ML과 오버레이 protoplasts은 설탕 기울기를 방해하지, 원심 분리기를 100 7 분, G.위한 </li><li> 효소 솔루션 및 W5 솔루션 (그림의 1B)의 인터페이스에서 protoplasts 10 ML를 수집하고 새로운 50 ML 팔콘 튜브로 전송할 수 있습니다. </li><li> 60 G.에서 5 분 15 ML W5 솔루션, 원심 분리기 추가 뜨는을 제거합니다. </li><li> 60g에서 5 분 대한 15ml W5의 솔루션, 원심 분리기에 protoplasts을 resuspending하여 효소 솔루션에서 무료 protoplasts 와시 </li><li> 1 - 3ml W5 솔루션에 뜨는, resuspend pelleted protoplasts를 제거합니다. </li><li> hemocytometer로 계산하여 세포 원형질의 수율을 평가해보십시오. </li></ol> 부 4. 시약 : TVL : sorbitol 0.3 M, 50 MM CaCL이 필터 - 소독 및 -20 ° C.에 저장 효소 솔루션 : 0.5 M의 자당, 10 MM MES - ​​코 [산도 5.7], 20 MM CaCl 2, 40 MM KCl, 1 % Cellulase (Onozuka R - 10), 1 %의 Macerozyme (R10). 필터 - 소독과 갓 사용합니다. W5 솔루션 : 0.1 % (W / V) 포도당, 0.08 % (W / V) KCl, 0.9 % (W / V) NaCl, 1.84 % (W / V) CaCl 2, 2 MM MES - ​​코 산도 5.7. 필터 - 소독하고 실온에서 저장합니다. 제 5 부 : 대표 결과 : 부품 1과 2에서 설명한 Arabidopsis 문화 조건을 사용하면 protoplasts (그림 1A)을 분리에 적합한 건강한 모종을 렌더링. 원형질은 자당 농도 그라디에 의해 정화 아르엔트 원심 분리 및 W5 및 효소 솔루션 (그림의 1B)의 인터페이스에서 수집했습니다. 제 3에 설명되어있는 원형질 분리 절차를 사용하여 우리는 일반적으로 신선한 모종 중 하나 그램에서 그대로 protoplasts의 10월 5일부터 10일까지 6 (그림 1C)를 수확. <img src="/files/ftp_upload/1149/Fig1.jpg" alt="그림 1" /> 그림 1. Arabidopsis 모종의 protoplasts의 고립. A, Arabidopsis 14 일 된 모종의 조직가의 수정 절차 (첸과 Halkier, 2000)이 수집하여 protoplasts로 변환되었습니다. B, Protoplasts은 자당 밀도 기울기 원심 분리에 의해 정화 및 효소 솔루션의 인터페이스에서 수집되었습니다 W5 uffer (화이트 화살표). C, protoplasts의 브라이트 - 필드 현미경. Microphotographs은 자이스 혈구 Axioscope이 플러스 현미경으로 연계 냉각 CCD 카메라를 사용하여 수집되었습니다.

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

식물 유전자의 기능 분석에의 이용에 대해이 절차를 설명 잠정 특허는 기술 기업 및 상용화에 대한 코넬 센터 (CCTEC) 월 2008 접수되었습니다. OK Vatamaniuk, 그리고 Zhai, 식물 유전자의 기능 분석에 대한 신속한 Z. 방법 : 내용 적요 대한 50341/015001 없습니다.

그대로 원형질의 높은 수율을 보장하기 위해서는 건강 식물 - 시작하는 것은 매우 중요하다. protoplasts를 분리 위해 필터 소독 솔루션을 사용합니다. protoplasts가되기 것을 기억하십시오. 따라서 protoplasts을 처리하는 경우, pipet 또는 그들을 브레이크 것입 적극적으로부터 와동을 혼합하지 않습니다. 대신 천천히 회전 또는 원심 튜브를 녹화하여 protoplasts를 섞는다. 적어도 96 H.에 대한 가능한 설명하?...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5524</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1296</td>
<td>&nbsp;</td>
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<td>Petri Dishes (100x15mm)</td>
<td>&nbsp;</td>
<td>Kreckeler Scientific</td>
<td>82-4001</td>
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<td>Cellulase (Onozuka R&#x2010;10)</td>
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<td>RPI Corp</td>
<td>C32200</td>
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<td>&nbsp;</td>
<td>RPI Corp</td>
<td>M22010</td>
<td>&nbsp;</td>
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<td>Cheesecloth wipes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>06-665-29</td>
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<td>Lab Rotator</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>1167152Q</td>
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<td>Allegra X&#x2010;15R Centrifuge</td>
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<td>VWR</td>
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<td>Axioskop2 Plus Microscope</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Protoplasts 자신의 세포 벽을 제거 효소가 있었 식물의 세포 수 있습니다. 다른 식물의 조직에서 protoplasts의 분리가 먼저 40 년 이상 전에보고되었다<sup&gt; 1</sup&gt; 이후 같은 subcellular 단백질의 지방화, 손상 organelles의 고립을 두 번 좌초 RNA 간섭 (RNAi)에 의해 유전자 불활 성화 대상으로 세포 프로세스 다양한, 연구에 적응되었습니다<sup&gt; 2-5</sup&gt;. 원형질 분리 프로토콜의 대부분은 성숙 Arabidopsis의 잎 조직 (예 : 35 일 된 식물)를 사용<sup&gt; 2-4</sup&gt;. 우리는 14 일 된 Arabidopsis의 모종을 채용하여 기존의 프로토콜을 수정했습니다. 이 절차에서는, 14 일 된 모종의 일g 5 10 굴복<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt; 최소한 96시간 그대로 유지 protoplasts. 모종의 protoplasts의 수확량은 성숙 Arabidopsis의 잎사귀에서 준비를 비교하지만, 대신 35-36일의 protoplasts의 분리는 15 일 이내에 완료됩니다. 이것은 성숙한 식물을 사용하는 경우 protoplasts를 분리하는 데 필요한 시간과 성장 챔버 공간을 감소 수 있으며 그대로 protoplasts을 필요로 하류 연구를 신속.

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

이 동영상은 Arabidopsis 14 일 된 모종의 조직에서 그대로 protoplasts를 분리하는 절차를 보여줍니다. 격리 protoplasts 적어도 96h 위해 그대로 유지하고 대신 한 달 된 성숙한 식물의 모종으로부터 격리되는 감안할 때,이 절차는 그대로 protoplasts가 필요한 assays을 신속.

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

isolation-protoplasts-from-tissues-14-day-old-seedlings-arabidopsis

Research

JoVE Journal

Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

36.0K 조회수.  Cornell University. 이 동영상은 Arabidopsis 14 일 된 모종의 조직에서 그대로 protoplasts를 분리하는 절차를 보여줍니다. 격리 protoplasts 적어도 96h 위해 그대로 유지하고 대신 한 달 된 성숙한 식물의 모종으로부터 격리되는 감안할 때,이 절차는 그대로 protoplasts가 필요한 assays을 신속.

비디오: Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

높은 처리량 원형질체 분리 및 변환을위한 로봇 플랫폼

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

연구

유전학

mRNA Interactome Capture from Plant Protoplasts

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called &#34;mRNA interactome capture,&#34; which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

식물 원형질에서 mRNA Interactome 캡처

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional ...

생화학

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

단백질 지 방화 Immunoblotting 또는 단백질의 분석에 대 한 애기에서 엽록체 하위 구획의 준비

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

Isolation of High Purity Tissues from Developing Barley Seeds

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.

Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

식물 Protoplasts의 형광 활성 세포 정렬

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

생물학

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

세포 유형 별 척도에서 애기장대 뿌리 개발을 조사하기 위해 리보솜 친화성 정제 (TRAP) 번역

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

발생학

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.

에서 외과를 이용한 셀 타입 특정 표현식 프로파일을 사용하여 조직 및 장기 특정 Phytochrome 응답 조사 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 ...

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.

에서 초기 단계의 배아의 효율적이고 신속한 분리<em&gt; 애기 장대</em&gt; 씨앗

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

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.

발광 시계 기자와 애기 장대 원형질체 트랜의 일주기 표현형의 신속한 분석

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

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

14 일 된 모종의 조직에서 Protoplasts의 분리 Arabidopsis thaliana

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