This work was supported by the Natural Sciences and Engineering Council of Canada (NSERC), the Canada Research Chair (CRC) program, and the Canadian Foundation for Innovation (CFI).

<html> 1. Bitki Kültürü ve Hazırlık <ol><li> (Ayrıntılar için, 10&#39;a bakınız) iklim-kontrollü bir büyüme odasında 7 gün boyunca arpa fide hidroponokal büyütün. NOT: Bu besin gereksinimleri yaş değişecek gibi, gelişim aşamaları çeşitli tesislerini inceleyerek dikkate almak önemlidir. </li><li> Bir gün deney öncesinde, tek bir çoğaltılmasına (DI için paket başına 3 bitkiler, Cate için paket başına 6 bitkiler) yapmak için birlikte birkaç fidan paket. Sürgünlerin taban kısmı etrafında Tygon hortumunun bir 2 cm&#39;lik bir parça sararak ve bir &quot;yaka&quot; oluşturmak için bant ile boru sabitlenmesiyle Paketi fideleri. NOT: Deste başına bitki sayısı deneysel koşullar 10,13,14 göre değişebilir. Bohçalama kök kütlesi ve / veya spesifik aktivite düşük, özellikle istatistik ve ölçüm doğruluğunu iyileştirmek için yapılır. </li></ol> Deneysel Çözümleri / Malzemelerin 2. Hazırlık içerik &quot;&gt; Not: Aşağıdaki tipik deney öncesinde 1 gün yapılır. <ol><li> Ve bitki materyali ve spesifik aktivite [S o için numune şişelere ((bitki örneklerinin spin-kurutma için), santrifüj tüpleri (11 ayrıntılar için bkz) Pre-etiketleme, etiketleme ve desorpsiyon çözümleri;: DI için aşağıdaki toplamak aşağıya bakınız]). Havalandırır ve tüm çözümleri karıştırın. </li><li> , (10 ayrıntılar için bkz) elüsyonların, bitki örnekleri için akış huniler, (bitki örneklerinin spin-kurutma için) santrifüj tüpleri ve numune şişeleri (Eh-karışık, gazlı etiketleme ve elüsyon çözümleri ve: Cate için aşağıdaki toplamak S o ve seyreltme faktörü belirlenmesi [D f; aşağıya bakınız]). </li></ol> 3. radyotracer hazırlayın DİKKAT: Aşağıdaki güvenlik adımlar radyoaktivite ile çalışma öncesinde alınmalıdır. <ol><li> Emin olun Radioac gereksinimleritif malzemeler lisans anlaşılır ve takip edilmektedir. Uygun güvenlik donanımları (yani, gözlük, eldiven, laboratuvar önlüğü, kurşun yelek / yaka) ve Dozimetreleri (örneğin, TLD halka ve rozet) giyin. Koruyucu Set up (yani, pleksiglas ve kurşun tuğla) ve bunun arkasında radyoaktif çalışmayı gerçekleştirmek. Geiger-Müller sayacı rutin kontaminasyon izlemek için mevcut olduğundan emin olun. </li><li> 42 K + hazırlanması <ol><li> Dengesi üzerinde temiz, kuru bir behere koyun. Dengesini sıfır. </li><li> Ambalajından (toz halinde 42 K 2 CO 3 20 mCi) izleyicinin şişesini çıkarın ve beher içine izleyici dökün. Kitle not alın. </li><li> Beher içine H 2 SO4, 0.07 ml, ardından dH 2 O Pipet 19.93 mi,. Bu aşağıdaki kimyasal reaksiyonu izlerler: 42 K 2 CO 3 (S) + H 2 SO 4 (l) + H 2 O (l) 42 → </sK 2&gt; kadar SO 4 (L) + CO 2 (v) + 2H 2 O (l) </li><li> K 2 CO 3 kütlesini ve moleküler ağırlık verilen radyoaktif stok çözeltisinin konsantrasyonu, ve hacim (20 mi) hesaplayın. NOT: 13 NH 3/13 NH4 + İzleyici suyun oksijen atomunun proton bombardımanı yoluyla bir siklotron üretilir çalışma halinde (tipik olarak 100-200 mCi faaliyet ile sonuçlanan, üretim detayları için, 12 bakınız). 14. NH 3/14 NH4 + &#39;nın miktarı, bu çözeltiler içinde son derece düşük olup, stok çözeltisi N içeriği ihmal edilebilir. </li></ol></li></ol> 4. Doğrudan Akın (DI) Ölçüm <ol><li> 42 K + için, etiketleme çözeltisi içine K + arzu edilen nihai konsantrasyonuna ulaşmak için gereken radyoaktif stok çözeltisi miktarı pipetle. <ol><li> NH 3/13 13 için NH 4 +, pipet etiketleme çözümü içine küçük bir miktar (&lt;0.5 ml). Etiketleme çözümü (havalandırması yoluyla) iyice karıştırmak için izin verin. </li></ol></li><li> Bir numune şişesi içine etiketleme çözeltisi, 1 ml alt-örnek pipet ve (toplam 4 örnekleri) ile üç kez tekrarlayın. <ol><li> Bir gamma sayacı kullanılarak, (&quot;dakika başına sayımları&quot;, cpm olarak) şişelere radyoaktivite ölçülür. (Bu gibi kısa ömürlü izleyiciler için özellikle önemlidir) sayaç cpm okumaları izotop çürüme için düzeltilmiştir şekilde programlanmış olduğundan emin olun. </li><li> Dört numune sayımları (CPM mi-1) ortalama ve (umol mi -1) çözeltisi içinde alt-tabaka konsantrasyonu ile bölmek suretiyle (CPM umol -1 olarak ifade edilen) sO hesaplayın. </li></ol></li><li> (Bakınız test koşulları altında bitkiler önceden dengeye gelmesi, 5 dakika boyunca ön-etiketlenmesi (radyoaktif olmayan) çözelti içinde kökleri daldırınÖrneğin, ön etiket zamanlı olarak varyasyonlar için 10,13,14). </li><li> 5 dakika için etiketleme (radyoaktif) çözümü kökleri daldırın. NOT: Etiketleme kez deney 3,4,7-10 göre değişiklik gösterebilir. </li><li> Yüzey-yapışan radyoaktivite uzaklaştırılması için, 5 saniye boyunca yayılım çözeltisine kökleri aktarın. Hücre dışı izleyici daha açık köklerine 5 dakika boyunca desorpsiyon çözeltisinden oluşan ikinci bir kap içine aktarın kökleri. </li><li> Ayrı sürgünler, bazal sürgünler ve kökler incelemek ve. </li><li> Düşük hızda 30 saniye boyunca santrifüj tüplerine kökleri ve spin örnekleri yerleştirin, klinik dereceli santrifüj (~ 5.000 xg) yüzeyini ve interstisyel su çıkarmak için. </li><li> Kökleri (taze ağırlık, FW) tartılır. </li><li> Bitki örneklerinde radyoaktivite sayımı (ateş, bazal sürgün ve kök; adım 4.2.1). </li><li> Akı hesaplayın. Aşağıdaki formül kullanılarak bitki içerisine akışını hesaplamak Φ = Q * / S o ağırlık L burada Φ akısı(Ľmol g -1 saat -1), Q * dokusunda biriken zerresinin miktarı (genellikle kök, sürgün, kopyaya, ve, kombine bazal sürgün), S o etiketleme çözümü (CPM ľmol spesifik aktivite - 1) w kök taze ağırlığı (g) &#39;dir ve t L etiketleme süresi (saat)&#39; dir. NOT: Daha sofistike hesaplama Cate elde parametrelere dayalı, etiketleme ve desorpsiyon sırasında köklerinden eşzamanlı izleyici akışı için hesap yapılabilir (, ayrıntılar için, 4 görmek aşağıya bakın). </li></ol> Tracer efflux (CATE) Ölçümü ile 5. komparmantal Analizi <ol><li> Etiketleme çözümü ve ölçü S o (- Yukarıdaki, 4.2 adımları 4.1) hazırlayın. </li><li> Seyreltme faktörü (D f) ölçün. Not: Genellikle, gama sayacında detektöre örnek nisbetle pozisyonu miktarını etkileyebilirradyasyon ölçülür. Ayrıntılar için tartışma bakın. <ol><li> SO ölçüldükten sonra, her bir örnek için H 2 O 19 ml ilave (örneğin, bu son hacim = yıkama sıvısı hacmi = 20 ml) eklenmiştir. Her 20 ml numune radyoaktiviteyi sayın (adım 4.2.1). </li><li> 20 ml&#39;lik numunelerin ortalama cpm olarak 1 mL&#39;lik numunelerin ortalama cpm bölünmesiyle D F hesaplayın. </li></ol></li><li> 1 saat etiketleme çözümü kökleri daldırın. </li><li> Tüm kök malzeme huni dahilinde sağlanması, huni akıttığı etiketleme çözümü ve transfer bitkilerden bitkileri çıkarın. Plastik yaka üzerine bant küçük bir şerit uygulayarak akış hunisi tarafına yavaşça güvenli bitkiler. </li><li> Yavaşça huni içine ilk eluatın dökün. Sayacını başlatmak (yukarı sayma). </li><li> Musluğunu açın ve 15 sn sonra numune şişesine eluatın toplamak (not: yıkama süresi değişir; aşağıya bakınız). Musluğunu kapatın. Yavaşça hunisi içine bir sonraki eluatın dökün. </li><li> Repeaİlk elde edilen nihai eluatına, aşağıdaki yıkama seri, geri kalan kısmı için bir adım 5.6 T: 15 saniye ile (dört kez), 20 saniye (üç kez), 30 saniye için (iki defa), 40 saniye (bir defa), 50 saniye (bir defa), 29.5 dakikalık bir elüsyon süresi toplam 1 dakika (25 kez), NOT: Sızdırma serisi deneysel koşullar 7-10,13,14 göre değişiklik gösterebilir. </li><li> Yıkama protokolü tamamlandıktan sonra, bitkiler hasat (- yukarıda 4.8, 4.6 adım). </li><li> (5.2, M f her elüatın için okuma çarpılması) gama sayacında yıkama sıvılarından ve bitki örneklerinde radyoaktivite sayısı. </li><li> Yıkama süresinin bir fonksiyonu olarak Arsa izleme atışının (cpm gr (kök FW) -1 dk -1). Kararlı durum koşulları için, doğrusal gerileme ve akılarının hesaplamaları, döviz yarılanma ömürleri ve havuz boyutları (detaylar için, 6-9 bakınız) gerçekleştirin. </li></ol>

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The authors have nothing to disclose.

Yukarıdaki örneklerde gösterildiği gibi, yöntem, in planta Radyoaktif besin ve toksik maddelerin tek yönlü akıyı ölçen bir güçlü bir araçtır. 1 NH3 akışı, belki de bir 225 umol g-1 st -1, aşan ulaşabileceği göstermektedir En iyi niyetli transmembran akı Hiç bir bitki sisteminde 13 bildirilmiştir, ancak net tozları ölçüldü, bu akının büyüklüğü görünür olmaz. NH3 büyük bir a...

Besin ve Toksik maddelerin alımı ve dağıtımı kuvvetle bitki büyümesini etkilemektedir. Buna göre, yatan taşıma süreçlerinin incelenmesi, özellikle beslenme optimizasyonu ve çevresel streslere bağlamlarda, bitki biyolojisi araştırma ve tarımsal bilimler 1,2 önemli bir alanı teşkil (örneğin, tuz stresi, amonyum toksisitesi). Bitkilerde akılarının ölçülmesi için yöntemler arasında baş önemli 1950&#39;lerde geliştirilmiş radyo-izotopik izleyiciler, kullanımı ve günümüzde yaygın olarak kullanılan olmaya devam etmektedir (3, örneğin, bakınız). Bu Mife (akı tahmini iyon mikroelektrot) ve SIET (tarama iyon seçici elektrot tekniği) ve kullanımı gibi dokularda kök orta ve / veya birikim, iyon seçici titreşimli mikroelektrot kullandığı için besin tüketme ölçümü gibi diğer yöntemler, İyon seçici floresanlı boyalar, ayrıca yaygın olarak uygulanır, ancak ağ grip tespit etmek için yetenekleri açısından sınırlıdırxes (örneğin, akışı ve dışarıya akış arasındaki fark). Radyoizotopların kullanılması, diğer taraftan, araştırmacı kinetik parametreleri çözmek için kullanılabilecek tek yönlü akıları izole etmek ve ölçmek için eşsiz bir yeteneği sağlar (örneğin, K, M ve V maks) ve kapasitesi hakkında bilgi sağlar, enerjisi, ulaşım sistemlerinin mekanizmaları ve düzenlenmesi,. Radyo-ile yapılan tek yönlü akış ölçümleri ters yönde akış kadar koşullar altında özellikle yararlı olan, hücre içi ve havuzlarının devir hızla 4&#39;tür. Takip izotop, aynı elemanın diğer bir izotopun bir arka planı görülmektedir çünkü Ayrıca, Radyoaktif yöntemleri, (aşağıda, &quot;Tartışma bakınız) ölçümlerinin bir çok diğer tekniklerin aksine, oldukça yüksek bir substrat konsantrasyonlarında iletilmesine olanak tanımaktadırlar. Burada, tek yönlü ve n radyoizotopik ölçümü için ayrıntılı adım sağlayansağlam Bitkilerde ve mineral besin tozları ve toksik maddeler. Vurgu akı potasyum (K +) ölçülmesi, bir bitki makro besinin 5 ve amonyak / amonyum yapılacaktır (NH3 / NH4 +), ancak, örneğin, yüksek konsantrasyonlarda (en mevcut olduğu zaman toksik olan başka bir makro besin, 1- 10 mM) 2. Bu radyoizotoplar 42 K + (t 1/2 = 12,36 saat) ve 13 NH 3/13 NH4 + (t 1/2 = 9,98 dakika), sırası ile, bir model sistem arpa sağlam fidelerinde (Hordeum vulgare L. kullanacak .), iki temel protokollerin açıklaması: tracer efflux (CATE) tarafından doğrudan akını (DI) ve kompartman analizi. Biz bu makalede sadece her protokol gerçekleştirmek için gerekli adımları açıklar başından itibaren dikkat etmelisiniz. Her tekniğin uygun, hesaplamalar ve teorinin kısa açıklamalar sağlanan, ancak ayrıntılı edilir Fuarlarımız&#39;S arka plan ve teori konuyla 4,6-9 birkaç önemli makalelerinde bulunabilir. Daha da önemlisi, bu protokoller diğer besin maddeleri / toksik maddelerin analizi akı genel olarak aktarılabileceği (örneğin, 24, Na +, 22, Na +, Rb + &#39;86, 13 NO 3 -) ve diğer bitki türlerine, birkaç uyarılar da olsa (aşağıya bakınız) . Biz de radyoaktif maddeler ile çalışan tüm araştırmacıların kurumun iyonizan radyasyon güvenliği regülatörü ile düzenlenmiş bir lisans altında çalışması gerekir önemini vurguluyoruz.

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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">Gamma counter</td>
			<td style="width:103px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Model: Wallac 1480 Wizard 3&#34;</td>
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			<td height="63" style="height:63px;width:216px;">Geiger-M&#252;ller counter</td>
			<td style="width:103px;">Ludlum Measurements Inc.</td>
			<td style="width:119px;"></td>
			<td>Model 3 survey meter</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">400-mL glass beakers</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-206</td>
			<td>For pre-absorption, absorption, and desorption solutions</td>
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			<td height="21" style="height:21px;width:216px;">Glass funnel</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89000-466</td>
			<td>For efflux funnel</td>
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			<td height="21" style="height:21px;width:216px;">Large tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">529297</td>
			<td>For efflux funnel</td>
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			<td height="21" style="height:21px;width:216px;">Medium tubing</td>
			<td style="width:103px;">VWR</td>
			<td align="right" style="width:119px;">684783</td>
			<td>For bundling</td>
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			<td height="21" style="height:21px;width:216px;">Small tubing</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">63013-541</td>
			<td>For aeration</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aeration manifold</td>
			<td style="width:103px;">Penn Plax Air Tech</td>
			<td style="width:119px;">vat 5.5</td>
			<td>To control/distribute pressurized air into solutions</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass scintillation vials</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">66022-128</td>
			<td>For gamma counting</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass centrifuge tubes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">47729-576</td>
			<td>For spin-drying root samples</td>
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			<td height="21" style="height:21px;width:216px;">Kimwipes</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470173-504</td>
			<td>For spin-drying root samples</td>
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			<td height="21" style="height:21px;width:216px;">Dissecting scissors</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470001-828</td>
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			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">470005-496</td>
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			<td height="42" style="height:42px;width:216px;">Low-speed clinical centrifuge</td>
			<td style="width:103px;">International Equipment Co.</td>
			<td style="width:119px;">76466M-4</td>
			<td>For spin-drying root samples</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144493</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette</td>
			<td style="width:103px;">Gilson</td>
			<td style="width:119px;">F144494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89079-470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10-mL pipette tips</td>
			<td style="width:103px;">VWR</td>
			<td style="width:119px;">89087-532</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analytical balance</td>
			<td style="width:103px;">Mettler toledo</td>
			<td style="width:119px;">PB403-S/FACT</td>
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measuring fluxes of mineral nutrients and toxicants in plants with radioactive tracers

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. Radioisotope tracing is a major technique used to measure such fluxes, both within plants, and between plants and their environments. Flux data obtained with radiotracer protocols can help elucidate the capacity, mechanism, regulation, and energetics of transport systems for specific mineral nutrients or toxicants, and can provide insight into compartmentation and turnover rates of subcellular mineral and metabolite pools. Here, we describe two major radioisotope protocols used in plant biology: direct influx (DI) and compartmental analysis by tracer efflux (CATE). We focus on flux measurement of potassium (K+) as a nutrient, and ammonia/ammonium (NH3/NH4+) as a toxicant, in intact seedlings of the model species barley (Hordeum vulgare L.). These protocols can be readily adapted to other experimental systems (e.g., different species, excised plant material, and other nutrients/toxicants). Advantages and limitations of these protocols are discussed.

Şekil 1 yüksek yetiştirilen sağlam arpa fidelerinin köklerine NH 3 akını (13 N) DI tekniği kullanılarak bulundu izotermlerini gösterir (10 mM) NH 4 + ve ya (0.02 mM), düşük veya yüksek (5 mM ) K +. (, Çözelti pH 13 değişim ile ayarlanır [NH3] dahili) NH3 tozlar dış NH3 konsantrasyonunun bir fonksiyonu olarak grafiğe zaman izotermleri Michaelis-Menten kinetiğine gösterir. NH 3 tozlar...

Watch this Scientific Journal Video about Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers at JoVE.com

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Radyoaktif izleyiciler ile Bitkilerde Mineral Besin ve Toksikantların Değişimler Ölçme

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

measuring-fluxes-mineral-nutrients-toxicants-plants-with-radioactive

Research

JoVE Journal

Environment

11.2K Görüntüleme.  University of Toronto. In planta measurement of nutrient and toxicant fluxes is essential to the study of plant nutrition and toxicity. Here, we cover radiotracer protocols for influx and efflux determination in intact plant roots, using potassium (K+) and ammonia/ammonium (NH3/NH4+) fluxes as examples. Advantages and limitations of such techniques are discussed.

Video: Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Rekombinant Cellulase Cel5A İfade itibaren<em&gt; Trichoderma reesei</em&gt; Tütün Bitkilerinde

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Çevre

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and &#8211;sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.

This manuscript describes how herbicide metabolism rates can be effectively quantified with excised leaves from a dicot weed, thereby reducing variability and removing any possible confounding effects of herbicide uptake or translocation typically observed in whole-plant assays.

Bir kesilmiş yaprak Testi ile Dikot Weeds Herbisit Metabolizma düşük Ölçme

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N2 is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N2:Ar ratios providing a means to measure N2 concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.

The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

Here, we present small core incubations for the measurement of sediment-water gas and solute exchange. These will provide reliable measurements of sediment-water exchange that assess the role of sediment in influencing biological and biogeochemical processes in aquatic ecosystems.

O Bentik Değişim<sub&gt; 2</sub&gt; K<sub&gt; 2</subKüçük Çekirdek KUluçkalar Kullanma&gt; ve Çözünmüş Besinler

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

Besin ve tortu tarım havza içinde sürekli Instream izleme

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Assessment of Methane and Nitrous Oxide Fluxes from Paddy Field by Means of Static Closed Chambers Maintaining Plants Within Headspace

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based on the diffusion theory. A known volume of air overlaying a defined soil area is enclosed within a parallelepiped cover (named &#34;chamber&#34;), for a defined period of time. During this enclosure period, gases (methane (CH4) and nitrous oxide (N2O)) move from soil pore air near their microbial source (i.e., methanogens, nitrifiers, denitrifiers) to the chamber headspace, following a natural concentration gradient. Fluxes are then estimated from chamber headspace concentration variations sampled at regular intervals throughout the enclosure and then analyzed with gas chromatography. Among the techniques available for GHG measurement, the static closed chamber method is suitable for plot experiments, as it does not require large homogenously treated soil areas. Furthermore, it is manageable with limited resources and can identify relationships among ecosystem properties, processes, and fluxes, especially when combined with GHG driving force measurements. Nevertheless, with respect to the micrometeorological method, it causes a minimal but still unavoidable soil disturbance, and allows a minor temporal resolution. Several phases are key to the method implementation: i) chamber design and deployment, ii) sample handling and analyses, and iii) flux estimation. Technique implementation success in paddy fields demands adjustments for field flooding during much of the cropping cycle, and for rice plant maintenance within the chamber headspace during measurements. Therefore, the additional elements to be considered with respect to the usual application of non-flooded agricultural soils consist of devices for: i) avoiding any unintended water disturbance that could overestimate fluxes, and ii) including rice plants within the chamber headspace to fully consider gases emitted through aerenchyma transportation.

The overall goal of this protocol is to measure greenhouse gas emissions from paddy fields using the static closed chamber technique. The measurement system needs specific adjustments due to the presence of both a permanent water layer in the field and of the plants within the chamber headspace.

Değerlendirme metan ve azot oksit Cerayanlar Paddy alanından aracılığıyla statik Headspace içinde bitki bakımı Chambers kapalı

This protocol describes the measurement of greenhouse gas (GHG) emissions from paddy soils using the static closed chamber technique. This method is based ...

Protocol for Producing Three-Dimensional Infrared Video of Freezing in Plants

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast make 2-dimensions (2D) infrared images somewhat difficult to interpret. Viewing an IR image or the video of plants freezing in 3 dimensions (3D) would allow a more accurate identification of sites for ice nucleation as well as the progression of freezing. In this paper, we demonstrate a relatively simple means to produce a 3D infrared video of a strawberry plant freezing. Strawberry is an economically important crop that is subjected to unexpected spring freeze events in many areas of the world. An accurate understanding of the freezing in strawberry will provide both breeders and growers with more economical ways to prevent any damage to plants during freezing conditions.
The technique involves a positioning of two IR cameras at slightly different angles to film the strawberry freezing. The two video streams will be precisely synchronized using a screen capture software that records both cameras simultaneously. The recordings will then be imported into the imaging software and processed using an anaglyph technique. Using red-blue glasses, the 3D video will make it easier to determine the precise site of ice nucleation on leaf surfaces.

Here, we present a protocol to image a strawberry plant freezing in 3 dimensions. Two infrared cameras positioned at slightly different angles are used to produce a red-blue anaglyph video to observe the freezing of the plant in 3 dimensions.

Bitkilerde buz gibi üç boyutlu kızılötesi Video üretimi için iletişim kuralı

Freezing in plants can be monitored using infrared (IR) thermography, because when water freezes, it gives off heat. However, problems with color contrast ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

Kök büyüme ve yerelleştirilmiş besin kavramaları görselleştirmek için en iyi duruma getirilmiş bir Rhizobox Protokolü

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

Kombine boyutu ve toprak yoğunluğu ayırma Organo-Mineral etkileşimlerin araştırmalar için

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ&#160;by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (&#60; ca. &#8722;0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2&#160;for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Observing the water distribution within the xylem provides significant information regarding water flow dynamics in woody plants. In this study, we demonstrate the practical approach to observe xylem water distribution in situ&#160;by using a cryostat and cryo-SEM, which eliminates artifactual changes in the water status during sample preparation.

Cryo-scanning Electron mikroskop ile görselleştirilen Woody bitkilerde xylem su dağılımı

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Toxicants Trans ve çok jenerasyonel etkileri okumak için Caenorhabditis elegans kullanma

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...