IEM은 자금 덴마크 연구위원회 (FTP 및 FNU)을 인정하고 싶습니다. ERL은 ARC DP 교부금의 지원을 인정합니다. AB는 식물 세포 벽의 부여에 우수의 ARC 센터의 지원을 인정한다.

1. 조직 수집 및 준비 <ol><li> 관심의 각 조직 최소한 세중의에 식물 조직의 100 밀리그램 신선한 무게를 (10 MG 건조 중량의 최소)를 수집합니다. 다음 단계는 식물 조직으로부터 세포 벽 재료의 준비에 대해 설명합니다. 저장 조직의 경우, 해제 - 원하는 녹말은 효소 이전에 13에 설명 된대로 세포 벽 고분자의 추출을하기 전에 제거됩니다. </li><li> 24 튜브 어댑터 세트와 3 밀리미터 텅스텐 카바이드 비즈 (30 Hz에서, 2 X 30 초)로, Qiagen TissueLyser II를 사용하여 액체 질소로 고급 분말로 샘플을 균질화. 몇 샘플을 처리하는 경우 또는 박격포와 유봉이 사용됩니다. </li><li> 10 ML 플라스틱 원뿔 튜브에 homogenates를 전송합니다. </li><li> RT 10 ML 80% 브이 / v 에탄올에 homogenates을 닦고 2 분 동안 힘차게 vortexing하여 세포벽 자료를 준비합니다. </li><li> 3500 XG에서 샘플을 원심 분리기와 표면에 뜨는 폐기. 표면에 뜨는 명확 때까지 70 % 에탄올 특히 엽록소를 포함하는 조직을 위해, 또는 적어도 세 번 씻어 반복합니다. </li><li> 100 % 아세톤과 최종 세척을 수행하고 밤새 공기 건조에 알코올 불용성 잔류 물 (AIR)를 포함하는 알약을 둡니다. </li><li> 체 공기 좋은, 균일 한 분말을 달성하고 제대로, 언젠가는 homogenate에 남아있는 지상 입자를 큰 제거하는 0.4 mm 2 메쉬와 샘플. </li><li> microtubes, 샘플의 혼합을 지원하기 위해 3mm 유리 구슬을 포함하는 각에 10 MG의 AIR 샘플 무게. </li></ol> 2. 세포 벽 Glycans의 추출 <ol><li> pectins과 관련된 Pectins, 그리고 고분자는 각 샘플의 50 μM C​​DTA (산도 7.5) 500 μl를 추가하여 샘플에서 추출됩니다. </li><li> 간단히 소용돌이 튜브는 용매는 샘플 소재와 접촉을 보장하고 8 Hz에서 3 시간 동안 TissueLyser를 사용하여 혼합합니다. </li><li> 주의 깊게 12,000 XG에서 샘플을 원심 분리기 연구4 ° C.에서 supernatants과 상점을 emove </li><li> 13,000 X g에서 남아있는 용매, 소용돌이와 원심 분리기를 희석 및 제거 할 드 - 이온화 물 1 ML에 알약을 씻으십시오. 펠렛을 방해하지 않으면이 단계를 반복하고 다음 단계로 진행하기 전에 튜브에서 모든 액체를 제거합니다. </li><li> 교차 연결 glycans는 순차적으로 0.1 %로 4M NaOH의 500 μl로 남아있는 세포 벽 펠렛에서 추출됩니다 단계 2.1에서 설명한 동일한 절차를 사용 w / v를 NaBH4 - CDTA 추출을 위해 2.4. NaBH 4함으로써 다당류의 필링베이스를 방지 알코올로 다당류의 감소 끝에 알데히드 (또는 케토) 그룹을 줄이기 위해 추가됩니다. </li><li> 원심 분리 후, supernatants는 다시 제거됩니다 4 ° C에 저장하고, 산탄은 다음 추출을하기 전에 드 - 이온화 물에 두 번 세척. </li><li> 선택적 단계로서, 이러한 셀룰로오스 등의 잔류 고분자는, 500 μl cadoxen (31 % V / V 1,2 - diami으로 압축이 풀립니다2.4 - 2.1 단계에 설명 된대로 동일한 절차를 사용하여 0.78 M CDO와 noethane). 또한, 남은 펠릿의 절대 셀룰로오스 내용은 아세트산 / 질소 assays을 (토론 참조)를 사용하여 결정될 수있다. </li></ol> 3. 인쇄 Microarrays <ol><li> 원심 분리기의 supernatants는 미립자를 제거 13,000 XG에서 세포 벽 고분자를 추출 포함하는. </li><li> 잘 폴리 프로필렌 384 샘플 조직 유형 및 추출 종류에 따라 배열되어 사전 정의 된 사용자 정의 레이아웃을 사용하여 microtiter 접시에 각 샘플의 50 μl를로드합니다. </li><li> 탈 이온수와 0, 5, 25 × 시리얼 희석 시리즈의 세포벽 폴리머 샘플을 희석. </li><li> 이러한 핀 높이 수집 및 시간을 머물러 있으며, 세척 단계와 같은 매개 변수가 microarrayer를 제어하는​​ 소프트웨어에 설정되어 있습니다. </li><li> 인쇄 챔버의 습도는 샘플 증발을 방지 60 %로 제어됩니다. </li><li> 인쇄 작업이 LabNEXT softwa를 사용하여 시작됩니다재와 microarray 레이아웃에 해당하는 프로그램입니다. </li><li> 로봇은 기계의 평면 판에 부착되어 20 X 20cm의 니트로 셀룰로스 막에 샘플 판에서 솔루션을 인쇄 할 모세관 채널 핀을 사용합니다. 배열의 각 지점이 솔루션의 15 NL를 포함하고 세중의에 인쇄됩니다. </li><li> 인쇄 작업이 완료되면 동일한 microarrays는 개별 배열에 막과 컷에 서로 옆에 인쇄됩니다. </li><li> 각 실험에서 배열을 더 많거나 적은 샘플, dilutions 또는 복제를 수용하기 위해 수정할 수 있습니다. </li></ol> 4. Glycan Microarrays의 프로빙 <ol><li> 인쇄 후, 비 특​​정 바인딩을 줄이기 위해 인산에 용해 w / V 무 지방 우유 분말은 2 시간 동안 실온에서 (MPBS) 생리 버퍼 5 %의 개별 microarrays를 차단합니다. </li><li> MPBS에서 2 시간에 세포 벽 glycan epitopes에 대한 특정 단클론 항체와 프로브 microarrays. 단클론 antib의 대부분세포 벽 glycans에 대한 오디 세 회사의 상업적 사용할 수 있습니다, Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ), Carbosource 서비스 ( <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> )와 PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ). </li><li> 부정적인 컨트롤 만 MPBS도없고, 1 차 항체와 incubated microarray를 포함합니다. </li><li> 인산 3 회가 아닌 특정 바인딩을 제거 할 5 분에 (PBS) 생리 버퍼 microarrays을 씻는다. </li><li> 2 시간에 MPBS에 고추 냉이 퍼 옥시 데이즈 (HRP)에 복합 이차 항체와 microarrays를 조사. 세포 벽 glycans에 대한 대부분의 단클론 항체 안티 마우스 또는 안티 쥐 보조 항체가 필요합니다. </li><li> 비 특정 바인딩을 제거하는 5 분에 3 회 PBS 버퍼로 세척 단계를 반복합니다. </li><li> chromogenic (3,3-Diaminobenzidine) 또는 chemil를 사용하여 microarrays를 개발uminecent (루미놀) 기판. </li></ol> 5. 양을 정함 <ol><li> 개발 후, 고해상도 (1,200 dpi의) 바탕 화면 스캐너를 사용하여 개별 microarrays를 검색 및 제외, 16 비트 TIFF 파일 (그림 3)로 이미지를 저장합니다. </li><li> 자동 격자 도구가 구비 이미지 처리 소프트웨어 (LabNEXT) Xplore 사용하여 각 영역의 핵심 강도를 계산할 수 있습니다. 통합 현장 강도는 각 지점을 둘러싼 그리드 영역에서 픽셀의 합계에서 파생됩니다. </li><li> 각 microarray에 대한 그리드 데이터는 txt 파일로 내보낼 수 있습니다 수동으로 분석을 위해 Excel 스프레드 시트로 가져올 수 있습니다. 온라인 도구 ( <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/가</a> ) 자동으로 번역 및 개인 txt 파일에서 데이터를 처리하기 위해 개발되었습니다. </li><li> 통합 현장 강도가 인쇄 복제에서 평균과 &#39;비열한 자리를 얻기 위해 dilutions 수 있습니다각 샘플에 대한 강도 &#39;값 (그림 2). 또한, 배열에 하나의 희석 가치에 상응하는 현물 신호는 각 샘플에 대한 상대적 glycan의 에피토프의 풍요 로움을 수량화하는 데 사용됩니다. </li><li> 다른 샘플 사이의 상대적인 평균 스폿 농도는 Excel 또는 온라인 heatmapper 도구 (에서 조건부 서식 사용하는 히트 맵 (그림 4)로 제공됩니다 <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm을</a> ). 각 항체 유형에 대한 데이터는 100 수정되어 5 %의 컷오프 값은 배경 신호 탐지를 제거하는 부과됩니다. </li></ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+cell+wall+polymers+during+antheridium+development+and+spermatogenesis+in+the+Charophycean+green+alga,+Chara.">The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara.</a> 2104, 1045-1056 (2009).</li><li>Singh, B., Avci, U., Eichler Inwood, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specialized+outer+layer+of+the+primary+cell+wall+joins+elongating+cotton+fibers+into+tissue-like+bundles.">A specialized outer layer of the primary cell wall joins elongating cotton fibers into tissue-like bundles.</a> Plant Physiol. 150, 684-699 (2009).</li><li>&#216;bro, J., S&#248;rensen, I., Derkx, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+of+Erwinia+chrysanthemi+pectin+methylesterase+variants+using+carbohydrate+microarrays.">High-throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays.</a> Proteomics. 9, 1861-1868 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

CoMPP는 단 몇일 만에 수백 개의 공장 파생 샘플 glycan 구성을 프로파일에 대한 신속하고 민감한 방법입니다. 이 방법은 lectins, 수용체, 그리고 항체 16와 같은 glycan 결합 단백질과 탄수화물의 상호 작용 높은 처리량 검사에 대해 이미 사용 가능한 박테리아 나 포유류의 glycan 배열 플랫폼을 보완합니다. 세포 벽 glycans를 검출하기위한 가능한 프로브의 큰 다양성으로,이 식물 세포 벽의 8,9?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 3mm의 텅스텐 카바이드 비즈 </td><td> Qiagen </td><td> 69,997 </td><td></td></tr><tr><td> 컬렉션 microtubes (1.2 mm) </td><td> Qiagen </td><td> 19,560 </td><td> 1.5 ML의 microfuge 튜브도 사용할 수 있습니다 </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85,300 </td><td></td></tr><tr><td> 3mm 유리 구슬 </td><td> 시그마 알드리치 </td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> 시그마 알드리치 </td><td> 34,588 </td><td></td></tr><tr><td> 산화 카드뮴 </td><td> 시그마 알드리치 </td><td> 202894 </td><td></td></tr><tr><td> 1,2 - diaminoethane </td><td> 시그마 알드리치 </td><td> 03,550 </td><td></td></<html> TR&gt; <tr><td> 니트로 셀룰로스 막 (0.22 μm의 기공 크기) </td><td> GE 물 및 공정 기술 </td><td> EP2HY00010 </td><td> 다른 기공 크기의 멤브레인은 다른 핀 유형에 적합합니다 </td></tr><tr><td> Xact II microarrayer 로봇 </td><td> Labnext </td><td> 001A </td><td> Xact II 로봇은 니트로 셀룰로스 막가 연결되어 할 수있는 사용자 정의 20 X 20cm 세라믹 플레이트가 장착 된 </td></tr><tr><td> RM microarray 핀을 Xtend </td><td> Labnext </td><td> 0037-350 </td><td> 핀은 멤브레인에 야생에 적합해야합니다 </td></tr><tr><td> 384 잘 microtiter 플레이트 (폴리 프로필렌) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> 안티 glycan 단클론 항체 </td><td> / 공장 프로브 CarboSource / Biosupplies </td><td></td><td> 웹 사이트, PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ), Carbosource (ww.carbosource.net &quot;대상 =&quot;_blank &quot;&gt; www.carbosource.net)와 Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> 안티 쥐 IgG (전체 분자) - 염소에서 생산 퍼 옥시 데이즈 항체. </td><td> 시그마 </td><td> A9037 </td><td> 이차 항체의 종류 (예 : 쥐, 마우스, 염소 등의 제기)를 사용하는 기본 항체에 따라 달라집니다. </td></tr><tr><td> 시그마 FAST 3,3 &#39;- Diaminobenzidine의 정제 </td><td> 시그마 </td><td> D4293 </td><td> 시약을 개발의 유형은 사용 보조 항체를 검출 방법 (colourmetric, 또는 chemiluminecent)에 따라 달라집니다. </td></tr><tr><td> SuperSignal 서 피코 Chemiluminescent 기판 </td><td> Thermoscientific </td><td> 34,080 </td><td> 위의 내용을 참조 </td></tr><tr><td> 이미지 처리 소프트웨어의 Xplore </td><td> LabNext </td><td> 008 </td><td> 자동 gridding 도구와 많은 소프트웨어 종류를 사용할 수 있습니다microarray 명소의 픽셀 값을 측정합니다. </td></tr><tr><td> 식물 다당류 </td><td> 시그마 / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

식물 세포의 벽은 생리학과 식물의 개발에 중요한 역할을하고 인간 사회 (예 : 나무, 종이, 섬유 및 바이오 연료 산업) 1,2의 원료를 제공 이기종 glycans의 복잡한 matrixes 있습니다. 그러나, 이러한 구성 요소의 생합성과 기능을 이해하는 것은 도전 남아있다. 세포 벽 glycans는 화학적 및 빌딩 블록, glycosyl 잔류의 복잡성으로 인해 conformationally 다양합니다. 이러한 형태의 여러 위치에서 연계 및 링 구조에서 차이가이 이성질체 또는 anomeric 구성 할 수 있으며,뿐만 아니라, 비 설탕 잔류의 배열로 대체됩니다. Glycan 구성도 다른 셀 및 / 또는 조직 유형 또는 단일 셀 벽 3의 하위 도메인에 따라 다릅니다. 또한, 자신의 구성은 개발 한 동안, 또는 환경 단서 사에 대한 응답으로 수정됩니다. 예에2000 유전자의 운은 식물 세포 벽 이기종 glycans의 복잡한 matrixes Arabidopsis 5 세포벽 glycan 생합성 및 수정에 참여 할 것으로 예상 했었 있습니다. 그러나, biosynthetic 유전자의 비교적 적은은 기능적으로 4,5 특징되었습니다. 유전자는 종종 differentially 세포 유형 6 시까 종종 낮은 수준에서 표현되기 때문에 역 유전학 방법이 어렵습니다. 또한, 돌연변이 연구는 종종 적절한 세포벽 기능이 7 유지하기 위해 유전자 중복이나 보상 메커니즘에 의해 방해되어 있습니다. 따라서 소설 접근 방식은 빠르게 glycan 구조의 다양한 범위를 특징하고 이해 세포벽 생합성 및 수정에 기능 유전체학 접근을 촉진하기 위하여 필요합니다. 단클론 항체 (mAbs)는 8,9 식물의 glycan 구조와 분포를 결정하는 중요한 도구로 부각되었습니다. 이러한 지구 인식pectins, xyloglucans, xylans, mannans, 글루칸과 arabinogalactans 등의 식물 세포의 벽 glycans의 주요 클래스, 내 현재 inct의 epitopes. 최근의 사용은 공장 및 조직 유형 동시에 9,10,11의 넓은 범위에서 glycans의 상대적 풍부한을 결정하기 위해 대규모 심사 실험으로 확장되었습니다. 여기 감소 시약 및 샘플 볼륨과 소형화 microarray 플랫폼을 사용하여 상영 할 (100 초) 여러 샘플을 수 종합 Microarray 폴리머 프로파일 (CoMPP) (그림 1 &amp; 2) 10,11라는 microarray 기반 glycan 검사 방법을 제시한다. microarray의 현장 신호 공식적으로 glycan 에피토프의 발생에 대한 반 정량 데이터를 제공하기 위해 정량화 할 수 있습니다. 이 방법은 잘 복잡한 생물학적 시스템 12 glycan 변경 사항을 추적하고 세포벽 성분의 글로벌 개요를 제공하는 데 적합합니다 특히 때 사전 지식 OF이 사용할 수 없습니다.

Nicotiana alata 꽃부터 6 조직 유형 (약의 필라멘트, 꽃가루, 난소, 꽃잎, sepals 및 오명)에서 glycans의 상대적인 풍요 로움은 CoMPP를 사용하여 결정되었다. 그림 3A은 부분적으로 (저) methylesterified homogalacturonan (HG), pectic 다당류 14 일 발생 에피토프에 대한적인 mAb JIM5 특정와 탐색 된 대표 microarray를 보여줍니다. JIM5 에피토프가 모든 꽃 조직의 CDTA 추출물에서 발견하는 것은 stigmatic 조직 <stron...

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

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

라고 기술<strong&gt; C</strong&gt; omprehensive<strong&gt; M</strong&gt; icroarray<strong&gt; P</strong식물 세포의 벽 glycans의 특성에 대한&gt; olymer 프로파일 (CoMPP)이 설명되어 있습니다. 이 방법은 생물학적 상황의 넓은 범위에서 glycan 발생의 검사를 허용하는 소형 microarray 분석 플랫폼으로 정의 glycan - epitopes로 이동 단클론 항체의 특이성이 조화를 이루고 있습니다.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

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

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

Research

JoVE Journal

Biology

14.5K 조회수.  University of Melbourne. 라고 기술 C omprehensive M icroarray P olymer 프로파일 (CoMPP)이 설명되어 있습니다. 이 방법은 생물학적 상황의 넓은 범위에서 glycan 발생의 검사를 허용하는 소형 microarray 분석 플랫폼으로 정의 glycan - epitopes로 이동 단클론 항체의 특이성이 조화를 이루고 있습니다.

비디오: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

Microarrays을 사용하여 세균의 유전자 발현 분석

연구

생물학

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

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

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

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

측정 식물 세포 벽 확장 (크리프)는 산성 산도에 의해 및 알파 Expansin에 의해 유도된

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

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

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

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

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

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 ...

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

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

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

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

Microarrays을 사용하여 tRNA의 Aminoacylation (충전) 레벨의 게놈 - 와이드 분석

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

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

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

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

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

세포외 다당류의 OLIgo 질량 프로파일 (OLIMP)

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

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

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

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

동위 원소 태그 질량 분광법을 사용하여 Thiol 산화 환원 프로테옴 프로 파일링

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

의 조직 화학 염색<em&gt; 애기 장대</em&gt; 차 전지 벽 요소

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

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

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

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

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

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

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

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

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

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

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

개별 세포 유형의 전사 프로파일 링을위한 도구로 레이저를 이용한 이외에 Microdissection (LAM)

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

An Easy Method for Plant Polysome Profiling

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

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

공장 Polysom​​e 프로파일 링을위한 쉬운 방법

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