우리는 그들의 기술 지원에 대 한 응용된 생물 과학 부, 도요 대학, 군마 농업 기술 센터의 씨 Koudai 타 니 구치의 씨 아키라 무라카미에 감사. 우리는 또한 그들의 제안에 대 한 교수 Shosaku Kashiwada 박사 우 Maheswari Rajagopalan, 도요 대학에 감사입니다. 이 연구는 생명 및 환경 과학, 도요 대학에 대 한 연구 센터에 의해 부분에서 지원 되었다.

참고: Ipecac (C. ipecacuanha)에 사용 되었다이 연구 생 phytohormones의 분석을 용이 하 게 하기 때문에.
1. 성장 조건 Ipecac의 우발적인 촬영 유도
<ol><li>PH 5.710, 조정 phytohormone 무료 B5 매체를 준비 하 고 0.2 %gellan 껌을 추가. 압력가 마로 소독 하 여 소독.</li>
	<li>멸 균 페 트리 접시 (90 m m × 20 m m)에 압력가 하는 매체의 25 mL를 붓으십시오.</li>
	<li>Ipecac plantlets 수술 메스를 사용 하 여 살 균 아크릴 플레이트에 블레이드 번호 22의 8 밀리미터 internodal 세그먼트를 잘라내어 매체 (그림 1)에 배치.</li>
	<li>24 ° C 15 µmol m−2의 − 1 5 주 동안 14 h 빛/10-h 어두운 주기에서 빛의 아래에 B5 매체 phytohormone 무료에 문화.</li>
	<li>지역 식별 난 (꼭대기) 4 (기저) 각 세그먼트 (그림 1B). 일주일에 한 번 우발적인 촬영 > 0.3 m m 길이 현미경으로 각 지역에서의 수를 계산 합니다.</li>
</ol>2. 추출 및 Phytohormones의 정화
<ol><li>각 4 개의 2 mL 샘플 튜브 5 m m 지 르 코니 아 비드를 넣어.</li>
	<li>지역으로 세그먼트를 잘라 iv (각 2 m m에 길이) 수술 메스를 사용 하 여 살 균 아크릴 판에 나.</li>
	<li>별도 샘플 튜브 (10-30 mg 신선한 무게)에 각 지역의 8 개 세그먼트를 수집 합니다.</li>
	<li>무게, 그리고 그 후 액체 질소에 샘플을 동결.</li>
	<li>비드 기반 균질 화기를 사용 하 여 얼음된 샘플을 짝사랑.</li>
	<li>Auxin의 각 내부 표준의 500 pg와 紀 (d5-IAA, d5tZ, d5tZR, d6-iP, d6-지적재산권)를 포함 하는 1 mL 이기에 짓 눌린된 샘플 일시 중지와 동 믹서를 사용 하 여.</li>
	<li>1 시간에 4 ° C에서 누른 다음 실 온에서 5 분 동안 3500 × g 에서 원심.</li>
	<li>1% (v/v) 초 산을 포함 하는 80% (v/v) 이기에 펠 릿을 세척. 원심 분리기 다시 3500 × g 에서 실 온에서 5 분. 일회용 유리 튜브에서 (2.7, 2.8 단계)에서 supernatants 결합.</li>
	<li>추가 600 µ L 각 결합된 상쾌한을 포함 1% (v/v) 초 산 물이 고 이기는 진공 집중 장치를 사용 하 여 증발.</li>
	<li>이기, 메탄올, 및 1% (v/v) 초 산을 포함 하는 물의 1 mL를 각각 적용 하 여 친수성 닥터지-평형 (HLB) 열 카트리지를 equilibrate.</li>
	<li>Equilibrated HLB 카트리지 당 하나의 샘플 솔루션을 적용 합니다.</li>
	<li>1 mL 물 1% (v/v) 초 산을 포함 하는 카트리지를 씻어.</li>
	<li>포함 하는 유리 튜브에 1% (v/v) 초 산 2 mL 80% (v/v) 이기와 모든 호르몬을 elute.</li>
	<li>포함 하는 1% (v/v) 초 산 진공 집중 장치를 사용 하 여 물에 추출 eluate에 이기를 증발. 참고:이 완전히 건조 하지 않습니다.</li>
	<li>1 mL 이기, 1 mL 메탄올, 0.5 mL를 적용 하 여 혼합 모드, 강한 양이온 교환 (MCX) 열 카트리지 equilibrate 0.1 M HCl, 그리고 포함 하는 1% (v/v) 초 산 1 mL 물.</li>
	<li>Equilibrated MCX 카트리지 당 하나의 샘플 솔루션을 적용 합니다.</li>
	<li>1 mL 물 1% (v/v) 초 산을 포함 하는 카트리지를 씻어.</li>
	<li>포함 하는 유리 튜브에 1% (v/v) 초 산 2 mL 30% (v/v) 이기와 IAA elute.</li>
	<li>씻어 카트리지 포함 하는 1% (v/v) 아세트산 2 mL 80% (v/v) 이기.</li>
	<li>씻으십시오 2 mL 물, 1 mL 물 5% 수성 암모니아를 포함 하는 카트리지.</li>
	<li>유리 튜브에 5% 수성 암모니아를 포함 하는 2 mL 60% (v/v) 이기와는 CKs을 elute.</li>
	<li>진공 농축 기를 사용 하 여 각 호르몬 분수의 용 매를 증발 하 고 LC-MS/MS 분석까지 −30 ° C에 저장 합니다.</li>
</ol>3. LC-MS/MS 분석 IAA 및 CKs
<ol><li>각 호르몬 추출 메탄올의 600 µ L 튜브에 녹이 고 나사 목 총 복구 유리병에 솔루션을 전송. 진공 집중 장치를 사용 하 여 용 매를 증발.</li>
	<li>20 µ L 30% (v/v) 이기, IAA 분수와 CK 분수 나사 목 총 복구 튜브에 1% (v/v) 초 산을 포함 하는 20 µ L 물에 용 해.</li>
	<li>HPLC 시스템 트리플 4 극 자 MS 시스템에서 긍정적인 이온 모드에서 샘플을 분석 합니다. 표 1에 나열 된 참고: 우리 HPLC 조건을 설정 합니다.<ol>
<li>IAA 차입에 대 한 5%-50% 용 매 B 7 분 이상의 이진 그라디언트를 사용 하 여 다음 98% 용 매 B에 의해 증가 1 분 동안 누른 다음 둥지를 주입 하 여 5% 용 매 B에 2 분 동안 equilibrate.</li>
		<li>CK 차입에 대 한 2%-40% 용 매 B 5 분 이상 40%-70% 용 매 B 7 분에서의 이진 그라디언트를 사용 하 여 다음 95% 용 매 B 늘려 1 분 동안 누른 다음으로 2% 용 매 B에 2 분 동안 equilibrate 주입을 중첩.</li>
		<li>분무 이온화 (ESI) 설정-MS 이온 소스 표 2에 나열 된 매개 변수.</li>
	</ol>
</li>
	<li>여러 반응 (MRM) 전환 표 3에 나열 된 각 분석의 정량화에 대 한 모니터링을 사용 합니다.</li>
	<li>계량 중수소 라는 표준 레이블 없는 비율의 표준 곡선에 대 한 내 생 IAA 및 CK 수준.</li>
</ol>

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Phytohormones의 분포를 식별 하기 위해 organogenesis에 관련 된 그것은 적용 될 때 phytohormones는 것 유도 위한 explants 때문에 있는 organogenesis phytohormone 무료 매체에 관찰 될 수 있다 식물 재료를 사용 하는 것이 중요 촬영 또는 뿌리, 그들은 전체 explant을, 세포 분화 및 organogenesis에 있는 식물의 고유한가 소성을 평가 하 고 어려운 영향을 미칩니다. 우발적인 촬영 phytohormone 무료 문화 미디어 Dianthus caryophy...

고트 Haberlandt (1854-1945 년) "totipotency", 어떤 식물에 의해 세포 수, 차별화, 분열과 성숙한 식물1에서 특정 세포 유형으로 차별화 그들의 이전 후에 전체 식물을 재생성의 개념을 제안 했다. 조직 문화에서 조합 및 성장 매체에 적용 된 것 phytohormones의 농도 의해 결정 됩니다 여부 식물 재생 여부 유도 될 수 있다. Skoog와 밀러 우발적인 뿌리 매체 포함 된 낮은 비율2에 유도 될 수 있는 반면 우발적인 촬영에 auxins, CKs의 높은 비율을 포함 하는 문화 매체에 굳은 살 담배에서에서 유도 될 수 있는 발견. 그 발견 이후 조직 문화는 널리 이용 된다 경제적으로 중요 한 작물의 전파 및 유전자 변형 식물3의 재생. 우발적인 촬영 촬영 정점 분열 조직, 잎, 뿌리, internodes 등 다른 조직에서 유도 될 수 있다. Phytohormone 치료는 대부분의 식물 종에서 우발적인 촬영의 유도 필요 합니다. 그러나, 최적 농도 조합 다 종에 의해 및 explants로 사용 하는 조직 중. 따라서, 많은 노력 결정 최적 농도 실험에 대 한 phytohormones의 조합으로 간다.
Carapichea ipecacuanha (Brot) L. 앤더슨 (ipecac)는 emetine 등 뿌리4에 주로 cephaeline, 알 카 로이드를 포함 하는 약용 식물 이다. 루트 추출 물 expectorant, 토 제, 그리고 amoebicide5로 사용 됩니다. 비록 ipecac 브라질의 열 대 우림에 자연적으로 성장, 문화, 씨앗을 설정 하기를 꺼 려 이며 발 아 율 감소는 추운 기후6일본, 씨앗 보관. 대신, 조직 문화에 의해 전파, 어떤 우발적인 촬영 internodes에 가장 효율적인 방법7,8. 흥미롭게도, 우발적인 촬영 phytohormone 치료8없이이 종에서 유도 될 수 있다.
우발적인 촬영 표 피 기저 지역9에 callusing, 없이 internodal 세그먼트의 꼭대기 지역에 형성 된다. 이 차이 아마도 phytohormonal 규정 internodal 세그먼트에서 조직 극성을 나타냅니다. Ipecac 문화 시스템 우발적인 촬영 형성 동안 내 생 phytohormone 레벨에 변화를 분석 하는 독특한 기회를 허용 합니다. 한 auxin (indole-3-아세트산 (IAA)) 및 4 개의 CKs 생 레벨의 분석에 대 한 우리의 방법을 소개 (isopentenyl 아데닌 (iP), isopentenyl 아데닌 아 (iPR), 트랜스-제 (tZ), 및 트랜스-제 아 (tZR)) 액정-MS/MS 사용 internodal 세그먼트.

<table><tbody><tr><td>[2H5]indole-3-acetic acid</td><td>Olchemlm Ltd</td><td>031 1531</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin</td><td>Olchemlm Ltd</td><td>030 0301</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H5]trans-zeatin riboside</td><td>Olchemlm Ltd</td><td>030 0311</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenine</td><td>Olchemlm Ltd</td><td>030 0161</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>[2H6]N6-isopentenyl adenosine</td><td>Olchemlm Ltd</td><td>030 0171</td><td>Internal standard for LC-MS/MS</td></tr><tr><td>indole-3-acetic acid</td><td>Wako</td><td>098 00181</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin</td><td>SIGMA-ALDRICH</td><td>Z0876 5MG</td><td>standard for LC-MS/MS</td></tr><tr><td>trans-zeatin riboside</td><td>Wako</td><td>262 01081</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenine</td><td>SIGMA-ALDRICH</td><td>D7674 1G</td><td>standard for LC-MS/MS</td></tr><tr><td>N6-isopentenyl adenosine</td><td>ACROS ORGANICS</td><td>22648 1000</td><td>standard for LC-MS/MS</td></tr><tr><td>acetonitrile hypergrade for LC-MS LiChrosolv</td><td>MERCK</td><td>1.00029.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>Water for chromatography LiChrosolv</td><td>MERCK</td><td>1.15333.1000</td><td>solvent for LC-MS/MS</td></tr><tr><td>HPLC</td><td>SHIMADZU</td><td>Prominence</td><td></td></tr><tr><td>MS</td><td>Sciex</td><td>3200QTRAP</td><td></td></tr><tr><td>Oasis HLB 30 mg/1 cc</td><td>Waters</td><td>WAT094225</td><td>cartridge column</td></tr><tr><td>Oasis MCX 30 mg/1 cc</td><td>Waters</td><td>186000252</td><td>cartridge column</td></tr><tr><td>screw neck total recovery vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td><td>Waters</td><td>186000274</td><td></td></tr><tr><td>Acquity UPLC BEH C18, 2.1x100 mm</td><td>Waters</td><td>186002350</td><td>UPLC column</td></tr><tr><td>Proshell 120 EC-C18, 2.1x50 mm</td><td>Agilent</td><td>699775-902</td><td>UPLC column</td></tr><tr><td>Digital microscope</td><td>Leica</td><td>DHS1000</td><td></td></tr><tr><td>TissueLyser II</td><td>QIAGEN</td><td>85300</td><td></td></tr><tr><td>Surgical blade</td><td>Feather</td><td>No. 22</td><td></td></tr><tr><td>Scalpel handle</td><td>Feather</td><td>No. 4</td><td></td></tr><tr><td>Savant SpeedVac/Refregerated vapor trap</td><td>Thermo Fisher Scientific</td><td>SPD111/RVT4104</td><td>vacuum concentrartor</td></tr><tr><td>Disposable glass tobe (13x100 mm)</td><td>IWAKI</td><td>9832-1310</td><td></td></tr><tr><td>Sterile petri dish</td><td>INA OPTICA</td><td>I-90-20</td><td></td></tr></tbody></table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

우발적인 촬영 형성 유전자 변형 식물의 재생 및 경제적으로 중요 한 작물의 전파에 대 한 중요 한 기술입니다. Phytohormone 치료는 대부분의 종에서 우발적인 촬영의 유도 필요 합니다. Auxin과 시 토 키 닌 (CK) 사이의 균형에 의해 결정 됩니다 여부 우발적인 촬영 유도 될 수 있는 수준. 많은 노력 결정 최적 농도 및 explants로 사용 하는 각 조직에 각 식물 종에 phytohormones의 조합으로 간다. 그러나 Ipecac에서, 우발적인 촬영 수 유도 될 phytohormone 치료 없이 문화 매체에서 internodal 세그먼트에. 평가할 셀 차별화 ipecac의 고유한 소성 수 있습니다. Ipecac 우발적인 촬영 유도, 우리 phytohormone 무료 B5 중간 5 주 동안 0.2 %gellan 껌으로 고형화에 14 h 빛/10-h 어두운 주기에 internodal 세그먼트 15 µmol m−2의 − 1 의 빛에서 24 ° C에서 경작. 우발적인 촬영 형성 동안 phytohormone 역학 조사, 우리 생 indole-3-아세트산 및 CKs 세그먼트에 의해 측정 액체 크로마토그래피 탠덤 질량 분석 LC-MS/MS. 이 메서드는 간단한 방식으로 생 indole-3-아세트산 및 CKs 레벨의 분석을 수 있습니다. 그것은 다른 식물 종에 organogenesis 동안 내 생 auxin과 CK의 역학 조사에 적용할 수 있습니다.

1세인트 주에서 아니 우발적인 촬영 형성 했다. 2노스다코타 주에 작은 쏘고 나타났다. 3rd , 4번째 주에 수 꼭대기 지역 (I 및 II)에서 주로 증가 쏘고 (그림 2A). 5일 주에 수 촬영의 되었고 약 7 시내 지역 나 지역 II (그림 2B) 5. 대조적으로, 단지 몇 가지 촬영 영역 II...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Ipecac의 위치인 문화 중 생 Auxin 및 시 토 키 닌의 정량화

우발적인 촬영 phytohormone 치료 없이 ipecac의 internodal 세그먼트에 유도 될 수 있다. 우발적인 촬영 형성 동안 phytohormone 역학을 평가 하기 위해 내 생 auxin 및 LC-MS/MS에 의해 internodal 세그먼트에서 시 토 키 닌을 측정 했습니다.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

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8.9K 조회수.  Toyo University. 우발적인 촬영 phytohormone 치료 없이 ipecac의 internodal 세그먼트에 유도 될 수 있다. 우발적인 촬영 형성 동안 phytohormone 역학을 평가 하기 위해 내 생 auxin 및 LC-MS/MS에 의해 internodal 세그먼트에서 시 토 키 닌을 측정 했습니다.

비디오: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

도구로 에틸렌 방출 화합물, 2- 클로로 에틸 산을 활용하여 박테리아의 에틸렌 응답을 공부하기

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

연구

생화학

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an Agrobacterium rhizogenes mediated transformation system on pigeon pea. The stem is infected with A. rhizogenes carrying a binary vector, which induced callus after 7 days and adventitious roots 14 days later. The generated transgenic hairy root was identified by morphological analysis and a GFP reporter gene.To further illustrate the application range of this system, CcCIPK14 (Calcineurin B-like protein-interacting protein kinases) was transformed into pigeon pea using this transformation method. The transgenic plants were treated with jasmonic acid (JA) and abscisic acid (ABA), respectively, for the purpose of testing whether CcCIPK14 responds to those hormones. The results demonstrated that (1) exogenous hormones could significantly upregulate the expression levelof CcCIPK14, especially in CcCIPK14 over-expression (OE) plants; (2) the content of Genistein in CcCIPK14-OE lines was significantly higher than the control; (3) the expression level of two downstream key flavonoid synthase genes, CcHIDH1 and CcHIDH2, were up-regulated in the CcCIPK14-OE lines; and (4) the hairy root transgenic system can be used to study metabolically functional genes in non-model plants.

CcCIPK14 Gene Function Analysis to Illuminate the Efficient Root Transgenic System

Here we present an efficient and stable transformation system for the functional analysis of the CcCIPK14 gene as an example, providing a technical basis for studying the metabolism of non-model plants.

An efficient and stable transformation system is fundamental for gene function study and molecular breeding of plants. Here, we describe the use of an ...

유전학

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant physiology. Due to their negative charge, low abundance and susceptibility to hydrolytic activities, the detection and quantification of these molecules is challenging. This is particularly the case for highly phosphorylated forms containing &#8216;high-energy&#8217; diphospho bonds, also termed inositol pyrophosphates (PP-InsPs). Due to its high sensitivity, strong anion exchange high-performance liquid chromatography (SAX-HPLC) of plants labeled with [3H]-myo-inositol is currently the method of choice to analyze these molecules. By using [3H]-myo-inositol to radiolabel plant seedlings, various InsP species including several non-enantiomeric isomers can be detected and discriminated with high sensitivity. Here, the setup of a suitable SAX-HPLC system is described, as well as the complete workflow from plant cultivation, radiolabeling and InsP extraction to the SAX-HPLC run and subsequent data analysis. The protocol presented here allows the discrimination and quantification of various InsP species, including several non-enantiomeric isomers and of the PP-InsPs, InsP7 and InsP8, and can be easily adapted to other plant species. As examples, SAX-HPLC analyses of Arabidopsis thaliana and Lotus japonicus seedlings are performed and complete InsP profiles are presented and discussed. The method described here represents a promising tool to better understand the biological roles of InsPs in plants.

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

SAX-HPLC를 사용하여 다른 식물 종에서 용해성, 방사성 라벨이 붙은 이노시톨 폴리인산염의 추출 및 정량화

The phosphate esters of myo-inositol, also termed inositol phosphates (InsPs), are a class of cellular regulators playing important roles in plant ...

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

특이적이고 효율적인 단백질 고갈을 달성하기 위한 튜닝 분해

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

면역학 및 감염병학

Lateral Root Inducible System in Arabidopsis and Maize

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is however tedious, because it occurs only in a few cells inside the root and in an unpredictable manner. To circumvent this problem, a Lateral Root Inducible System (LRIS) has been developed. By treating seedlings consecutively with an auxin transport inhibitor and a synthetic auxin, highly controlled lateral root initiation occurs synchronously in the primary root, allowing abundant sampling of a desired developmental stage. The LRIS has first been developed for Arabidopsis thaliana, but can be applied to other plants as well. Accordingly, it has been adapted for use in maize (Zea mays). A detailed overview of the different steps of the LRIS in both plants is given. The combination of this system with comparative transcriptomics made it possible to identify functional homologs of Arabidopsis lateral root initiation genes in other species as illustrated here for the CYCLIN B1;1 (CYCB1;1) cell cycle gene in maize. Finally, the principles that need to be taken into account when an LRIS is developed for other plant species are discussed.

Lateral Root Inducible System in Arabidopsis and Maize

The Lateral Root Inducible System (LRIS) allows for synchronous induction of lateral roots and is presented for Arabidopsis thaliana and maize.

의 측면 루트 유도 성 시스템<em&gt; 애기 장대</em&gt;과 옥수수

Lateral root development contributes significantly to the root system, and hence is crucial for plant growth. The study of lateral root initiation is ...

생물학

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

전략은 트로픽 응답에 Callose 매개 Plasmodesmal 게이팅의 역할을 확인하려면

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

식물 / 미생물 분자 상호 작용 및 신호의 동시 및 체계적 분석을위한 수경 공동 공동 배양 시스템

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

격리 된 괴 경 Protoplasts의 Cytometry에 의해 측정 Endoreduplication

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

표피 표현형 점수를 통한 기공 발달에 관여하는 유전자 식별

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Arabidopsis 묘목에서 엽록소 생합성의 고해상도 이미징

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

저자 스포트라이트: De-etiolation 중 엽록소 합성의 비침습적 고분해능 측정

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

애기장대 탈에티올화의 초기 단계 동안 엽록소 생합성 역학 측정을 위한 비침습적 분석

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

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