这项工作得到了以色列农业部首席科学家 (#16-16-0003) 的赠款的支持, 得到了以色列国科学、技术和 Spaceand。

1. 植物材料和生长条件
<ol><li>种子一烟草 (品种萨姆松) 种子每 5 cm x 5 cm 罐充满标准罐培养基。把花盆放在托盘上。在长日条件下的生长室中种植植物 (16/8 小时光/暗) 在恒温23摄氏度。用自来水灌溉, 直到水从锅里排水。</li>
	<li>50±5天后, 根据适宜的试验浓度, 开始在灌溉中进行铁处理。例如, 我们使用了一系列的铁浓度从0到6毫米, 辅以可溶性铁离子螯合剂 (fe EDDHA)。每两天用适当的溶液灌溉植物 (以避免脱水) 6-8 天。</li>
</ol>2. 准备用于铁测量的叶子
注: 所有要使用的材料必须是无铁的, 以减少铁污染的风险.用 4% HCl 溶液清洗砂浆和杵两次, 每次使用前用过滤纸干燥。如果任何材料被重用, 用 4% HCl 溶液清洗两次, 用滤纸干燥。
<ol><li>使用手套 (不要使用任何金属设备) 用手将叶子从茎上分离出来。每个样品使用约10克的叶子 (新鲜重量)。使用喷雾瓶清洁每片叶片的双蒸馏水 (DDW)。这一步骤对于避免铁污染是很重要的。</li>
	<li>把纸巾上的叶子晒干, 放进纸袋里。将纸袋转移到烤箱, 在恒温80摄氏度2-3 天。</li>
	<li>干燥时, 用砂浆和杵将叶子粉碎, 转到无菌的15毫升塑料管。</li>
</ol>3. 把树叶烧成灰烬
注: 使用低 pH 值 (接近 0) 的盐酸溶液的目的是提高铁的溶解度。岩石羊毛用于防止气体在燃烧过程中逃逸。
<ol><li>称一个新的, 密封的20毫升闪烁瓶没有它的盖子。使用 "包装" 按钮记下该值或将值设置为零。将粉碎干燥的叶子 (样品) 添加到瓶子里。</li>
	<li>权衡样品和容器并记下值。用岩石羊毛把瓶子关上。</li>
	<li>在不添加样品的情况下重3瓶, 并记下它们的值。这些小瓶将被用来作为控制, 以评估岩石羊毛的数量, 可能导致任何增加样品重量。</li>
	<li>将样品和控制瓶放在炉子里, 用以下温度步骤开始燃烧: 室温, 快速增加到425摄氏度, 最后, 425°C 4 小时。到了这个时候, 干树叶就会变成灰烬。</li>
	<li>让样品冷却到约100摄氏度, 但不低于这个温度以下两个步骤, 以避免湿度, 这可能会影响到样品的最终重量。使用重型手套, 从炉内取出带镊子的样品, 拿着瓶子 exteriorly。</li>
	<li>把瓶子放在平坦的表面上, 取下岩石羊毛, 用原来的盖子把瓶子关上。</li>
	<li>称3控制瓶 (见 3.3) 并计算其平均重量增益。如果重量增益等于或高于1% 的灰分重量 (见步骤 4.2), 请使用此值作为测量误差的估计。</li>
</ol>4. 为铁的测量准备灰烬
注: 在初始样品中的最终铁浓度计算为灰烬的重量除以盐酸的增加量。
<ol><li>在1米 hcl 溶液 (4% hcl) 中加入12.5 毫升的 37% hcl 溶液, 以87.5 毫升的 DDW (在塑料或玻璃瓶)。</li>
	<li>重量15毫升塑料管, 并记下值或设置值为零使用 "包装" 按钮。把骨灰转移到管子上, 权衡, 并记下值。这是灰的重量。</li>
	<li>在灰烬中加入5毫升的1米 HCl。过滤灰烬通过一个22µm 过滤器, 并添加一个额外的5毫升1米 HCl 通过同一过滤器。</li>
	<li>最后的音量应该是10毫升。请注意, 该解决方案的一部分将在筛选器中丢失。 注: 样品现在可以用火焰原子吸收光谱法或普鲁士蓝方法进行 Fe 测量。</li>
	<li>用原子吸收光谱法和普鲁士蓝方法 (见图 4) 测定每种植物的铁浓度, 制作一个标定曲线。随后, 仅用普鲁士蓝法测量铁浓度。</li>
</ol>5. 火焰原子吸收光谱法测定铁浓度
<ol><li>用火焰原子吸收光谱法从每个样品中去除4毫升。</li>
	<li>将火焰原子吸收光谱法测量所得的结果除以灰烬的重量。将结果值除以 0.01 (因为骨灰在10毫升可溶性)。产生的值是每克灰分 (ppm) 的铁浓度。</li>
</ol>6. 准备普鲁士蓝染色液
<ol><li>通过添加4克4Fe (CN)6到100毫升 DDW 和涡旋 (其他体积和/或浓度可用于不同的要求), 准备4% 普鲁士蓝溶液。应该指出的是, 在这项研究中, 使用了比以前报告 (20%)14更少浓缩的普鲁士蓝溶液。</li>
	<li>保持在黑暗中的解决方案在4°c, 直到使用。该解决方案是稳定的6月时, 储存在这样的条件。</li>
</ol>7. 使用火焰原子吸收光谱法生成普鲁士蓝方法的校准曲线
注: 用以下公式计算灰烬中铁的浓度 <img alt="Equation" src="/files/ftp_upload/57408/57408eq1.jpg">等式1 C: 浓度, V: 样品体积, W: 灰分重量 (g)。
<ol><li>混合0.50 毫升普鲁士蓝溶液和0.50 毫升1米 HCl。这将作为空白解决方案。</li>
	<li>混合0.5 毫升的样品 (骨灰在 4% HCl, 如3节所述) 和0.5 毫升普鲁士蓝溶液 (步骤 6.1) 由吹打。至少等待1分钟, 但不超过5分钟。5分钟后, 样品中的沉淀会发生。</li>
	<li>使用分光光度计将混合体转移到试管并测量 715 nm 的 OD。记下该值。</li>
	<li>将 OD 值 (步骤 7.3) 除以样品的灰重 (步骤 3.2)。结果表示每克灰的 OD。</li>
	<li>绘制从火焰原子吸收光谱测量 (Y 轴) 和 OD 值 (X 轴) 获得的铁浓度之间的线性回归。使用步骤5.2 和7.4 中获得的结果。计算回归公式, y = a +, y 代表铁浓度, a 代表吸光度相交, b 代表吸光度斜率, X 代表 OD。</li>
</ol>8. 使用普鲁士蓝法测定同一植物类型的其他样品中的铁含量
注: 由于这种类型的植物已经建立了校准曲线, 所以从同一植物类型的任何新样品中的铁浓度都可以用线性回归公式直接计算出来。
<ol><li>执行3和4节中的步骤, 后跟步骤7.1 到7.4。</li>
	<li>用线性回归 (步骤 7.5) 得到的公式计算溶液中铁的浓度。</li>
</ol>

<ol>
	<li>Kobayashi, T., Nishizawa, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+uptake,+translocation,+and+regulation+in+higher+plants.">Iron uptake, translocation, and regulation in higher plants.</a> Annual Review of Plant Biology. 63 (1), 131-152 (2012).</li><li>Bradley, J. M., Le Brun, N. E., Moore, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferritins:+Furnishing+proteins+with+iron.">Ferritins: Furnishing proteins with iron.</a> Journal of Biological Inorganic Chemistry. 21 (1), 13-28 (2012).</li><li>Zieli&#324;ska-Dawidziak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+ferritin+-+a+source+of+iron+to+prevent+its+deficiency.">Plant ferritin - a source of iron to prevent its deficiency.</a> Nutrients. 7 (2), 1184-1201 (2015).</li><li>Johnson, D. C., Dean, D. R., Smith, A. D., Johnson, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function,+and+formation+of+biological+iron-sulfur+clusters.">Structure, function, and formation of biological iron-sulfur clusters.</a> Annual Review of Biochemistry. 74 (1), 247-281 (2015).</li><li>Guo, H., Barnard, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+iron+oxide+nanoparticles:+morphology,+surface+chemistry+and+environmental+stability.">Naturally occurring iron oxide nanoparticles: morphology, surface chemistry and environmental stability.</a> Journal of Materials Chemistry A. 1 (1), 27-42 (2013).</li><li>Hem, J. D., Cropper, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+iron+in+natural+water.">Chemistry of iron in natural water.</a> Report US Geological Survey. , 1-31 (1962).</li><li>Rout, G. R., Sahoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+iron+in+plant+growth+and+metabolism.">Role of iron in plant growth and metabolism.</a> Reviews in Agricultural Science. 3, 1-24 (2015).</li><li>Speretto, R. A., Ricachenevsky, F. K., Stein, R. J., de Abreu Waldow, V., Fett, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+stress+in+plants+Dealing+with+deprivation+and+overload.">Iron stress in plants Dealing with deprivation and overload.</a> Plant Stress. 4, 57-69 (2010).</li><li>Tautkus, S., Steponeniene, L., Kazlauskas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+iron+in+natural+and+mineral+waters+by+flame+atomic+absorption+spectrometry.">Determination of iron in natural and mineral waters by flame atomic absorption spectrometry.</a> Journal of the Serbian Chemical Society. 69 (5), 393-402 (2006).</li><li>Braunschweig, J., Bosch, J., Heister, K., Kuebeck, C., Meckenstock, R. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reevaluation+of+colorimetric+iron+determination+methods+commonly+used+in+geomicrobiology.">Reevaluation of colorimetric iron determination methods commonly used in geomicrobiology.</a> Journal of Microbiological Methods. 89 (1), 41-48 (2012).</li><li>PerkinElmer. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atomic Spectroscopy - Guide to Selecting the Appropriate Technique and System. 16, (2011).</li><li>Wachasunder, S. D., Nafade, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+and+accuracy+control+in+the+determination+of+heavy+metals+by+atomic+absorption+spectrometry.">Precision and accuracy control in the determination of heavy metals by atomic absorption spectrometry.</a> Science. 58, 517-528 (2001).</li><li>Woods, J. T., Mellon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thiocyanate+method+for+iron.+A+spectrophotometric+study.">Thiocyanate method for iron. A spectrophotometric study.</a> Industrial &amp; Engineering Chemistry Analytical Edition. 13 (8), 551-554 (1941).</li><li>Perls, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nachweis+von+Eisenoxyd+in+gewissen+Pigmenten.">Nachweis von Eisenoxyd in gewissen Pigmenten.</a> Virchows Archiv Fur Pathologische Anatomie Und Physiologie Und Fur Klinische Medizin. 39 (1), 42-48 (1867).</li><li>Connorton, J. M., Jones, E. R., Rodriguez-Ramiro, I., Fairweather-Tait, S., Uauy, C., Balk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+expression+of+a+vacuolar+iron+transporter+doubles+iron+content+in+white+wheat+flour.">Altering expression of a vacuolar iron transporter doubles iron content in white wheat flour.</a> bioRxiv. , 1-25 (2017).</li><li>de la Fuente, V., Rufo, L., Rodr&#237;guez, N., Franco, A., Amils, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+iron+localization+in+wild+plants+and+hydroponic+cultures+of+Imperata+cylindrica+(L.)+P.+Beauv.">Comparison of iron localization in wild plants and hydroponic cultures of Imperata cylindrica (L.) P. Beauv.</a> Plant Soil. 418 (1-2), 25-35 (2017).</li><li>Hsiao, P. Y., Cheng, C. P., Koh, K. W., Chan, M. 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+Arabidopsis+defensin+gene,+AtPDF1.1,+mediates+defence+against+Pectobacterium+carotovorum+subsp.+carotovorum+via+an+iron-withholding+defence+system.">The Arabidopsis defensin gene, AtPDF1.1, mediates defence against Pectobacterium carotovorum subsp. carotovorum via an iron-withholding defence system.</a> Science Reports. 7 (1), 1-14 (2017).</li><li>Johnson, D. B., Kanao, T., Hedrich, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+transformations+of+iron+at+extremely+low+pH:+Fundamental+and+applied+aspects.">Redox transformations of iron at extremely low pH: Fundamental and applied aspects.</a> Frontiers in Microbiology. 3, 1-13 (2012).</li><li>Stumm, W., Lee, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygenation+of+ferrous+iron.">Oxygenation of ferrous iron.</a> Industrial &amp; Engineering Chemistry. 53 (2), 143-146 (1961).</li><li>Jones, A. M., Griffin, P. J., Collins, R. N., Waite, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferrous+iron+oxidation+under+acidic+conditions+-+The+effect+of+ferric+oxide+surfaces.">Ferrous iron oxidation under acidic conditions - The effect of ferric oxide surfaces.</a> Geochimica et Cosmochimica Acta. 145, 1-12 (2014).</li><li>Hawkesworth, C. J., Kemp, A. I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+continental+crust.">Evolution of the continental crust.</a> Nature. 443 (7113), (2006).</li><li>Thompson, L. M., Louis, M., Troeh, F. R., Thompson, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soils and soil fertility. , (1973).</li><li>Krueger, B. J., Grassian, V. H., Cowin, J. P., Laskin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+chemistry+of+individual+mineral+dust+particles+from+different+dust+source+regions:+The+importance+of+particle+mineralogy.">Heterogeneous chemistry of individual mineral dust particles from different dust source regions: The importance of particle mineralogy.</a> Atmospheric Environment. 38 (36), 6253-6261 (2004).</li><li>Bewick, V., Cheek, L., Ball, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistics+review+7:+Correlation+and+regression.">Statistics review 7: Correlation and regression.</a> Journal of Critical Care. 7 (6), 451-459 (2003).</li><li>Asuero, A. G., Sayago, A., Gonz&#225;lez, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+coefficient:+An+overview.">The correlation coefficient: An overview.</a> Critical Reviews in Analytical Chemistry. 36 (1), 41-59 (2006).</li><li>. Analytical Chemistry. Calibration Curves Available from: <a target="_blank" href="https://www.jove.com/science-education/10188/calibration-curves">https://www.jove.com/science-education/10188/calibration-curves</a> (2018)</li></ol>

作者没有什么可透露的。

植物组织中铁的测量对于评价灌溉或其他环境条件的影响非常重要。在这里, 我们描述了一个简单和准确的比色法测定烟叶中的铁含量, 可以很容易地适应其他植物种类和组织。
在优化比色法的条件下, 我们用低 ph 介质 (ph < 1.0) 来允许铁的溶解度。燃烧过程的执行, 以释放所有形式的铁, 并确保不存在的污染物在样品将修改结果。关于铁污染, 人们应该记住, 6.7% 的地壳包括氧化...

铁 (Fe) 是所有生物中重要的微量营养素。在植物中, 它是一种必需的微量营养素1 , 因为它参与了基本的过程, 如呼吸, 光合作用和叶绿素生物合成。由于反应导致释放自由基导致氧化应激, 游离铁离子的高积累对植物细胞有害。为了维持植物细胞内的铁稳态, 离子储存在 ferritins 内, 直接参与铁稳态2的蛋白质笼和所有生物体中铁的主要贮存结构。同时, 缺铁性贫血会影响到人口的很大比例, 从而增加了对植物铁生物强化的需求。由于植物铁蛋白的独特特性, 用铁蛋白进行食品浓缩, 为解决营养不良3的问题提供了一个有前景的战略。
铁离子主要见于两种氧化状态, 即黑色金属 (价 fe2 +或铁 (II)) 和铁 (三价 fe3 +或铁 (III)) 的形式。在细胞中也发现了其他几种形式的铁, 如铁簇4。Fe 在细胞之内被存放作为氧化铁并且自然地形成 hematites (fe2O3) 和 ferryhidrites (fe3 +)2O3· 0.5H2o) 在生理情况下5。在这些反应中形成的氢氧化物, 特别是铁形态, 具有很低的溶解度。铁的保留因此受到溶液 ph 值的影响, 并且在很大程度上是在 ph 值 56以上的固态状态。
考虑到铁的溶解度低、反应性高, 其在植物组织和器官中的转移必须与合适的螯合分子相联系。此外, 它的氧化还原状态之间的铁和铁形式1必须控制。在叶内, 约80% 的铁是在光合细胞中发现的, 由于其在电子运输系统中的重要作用, 在生物合成细胞色素, 叶绿素和其他血红素分子, 以及在形成铁 S 簇7。在细胞内铁过量的情况下, 剩余移位进入液泡中, 金属在铁蛋白分子中储存8。
在植物组织中, 铁可以用几种方法来测量, 包括火焰原子吸收光谱9 (原子吸收光谱法) 或比色测定10, 前者比后者精确得多。火焰原子吸收光谱法是一种高度精确的技术, 使人们能够根据单个元素的电磁辐射来确定样品的元素组成。火焰原子吸收光谱法将金属离子转化为原子态, 通过样品的加热, 导致离子激发和特定波长的发射, 当给定离子返回其基态时。不同离子的排放由单色仪分离, 并通过吸收传感器11进行检测。火焰原子吸收光谱因而服务直接地定量铁浓度。然而, 在生物组织中可视化铁的其他技术是可用的。电感耦合等离子体质谱 (ICP)12是一种非常精确的测量铁和其他微量元素的技术, 但对于火焰原子吸收光谱和 ICP-ms 的设备缺乏是一个普遍的问题。另一方面, 硫氰酸盐比色法测定铁量13缺乏精度, 无法检测样品之间的细微变化。普鲁士蓝染色14、15、16、17是基于铁离子盐 (K4Fe (CN)6) 反应的间接方法, 产生了强蓝色, 用于动物和植物组织组织学切片中的定性铁检测。
金属 (零价) 铁在岩石圈中是罕见的。环境中铁的主要非复合离子形式主要由环境中的氧量决定, 铁在缺氧环境中相对丰富, 铁在有氧场所占主导地位。后一种形态在极酸性环境中也占主导地位, 但在缺氧和酸性环境下, 铁氧化的致病剂往往不同于18。当铁在 4% HCl (pH 0) 在一个有氧环境中可溶性时, 稀释铁的主要部分存在作为铁形式 (Fe3 +)19,20。
铁离子与 K4Fe (CN)6的反应如下:
Fe3 +: FeCl3 + K4Fe (cn)6 = KFe (III) Fe (cn)6¯ + 3KCl
Fe2 +: 4 FeCl2 + 2 K4Fe (cn) 6 = fe4(Fe (CN)6)2 + 8 氯化钾
在本研究中, 我们询问普鲁士蓝染色是否有助于测定溶液中的铁含量。
初步验证了水溶液中铁浓度与普鲁士蓝染色的相关性。Fe (作为 FeCl2, FeCl3或1:1 混合物的二) 集中在水溶液由原子吸收光谱和吸收 (OD) 在普鲁士人的蓝色的加法以后测量。图 1显示了每个方法获得的测量的线性回归曲线。结果表明, 普鲁士蓝法可用于定量分析溶液中铁的浓度。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57408/57408fig1.jpg"> 图 1: 用火焰原子吸收光谱法测定的铁浓度与由普鲁士蓝方法获得的光吸收 (OD, 715 nm) 之间的线性回归.蓝色正方形和线代表 fe2 +解答, 红色正方形和线代表 fe3 +解答, 并且黑正方形和线代表1:1 混合物在 fe2 +和 fe3 +之间。得到了以下回归: [Fe2 +] = 3 + 123 x OD, r = 0.996, r2 = 0.989;[Fe3 +] = 1 + 292 x OD, r = 0.999, r2 = 0.997;和 [Fe2 +/3 +] = 11 + 146 x OD, r = 0.983, r2 = 0.956。fe2 +捐赠者是 FeCl2和 fe3 +捐赠者是 FeCl3。<a href="https://www.jove.com/files/ftp_upload/57408/57408fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了适应比色普鲁士蓝法对植物组织进行定量分析, 用火焰原子吸收光谱和普鲁士蓝染色测定烟叶灰分的铁含量。这两种技术的结果有很好的相关性。

<table>
	<tbody>
		<tr>
			<td>Potassium Hexacyanoferrate(II)</td>
			<td>Fisher Chemical</td>
			<td>14459-95-1</td>
			<td>Reagent for the Pussian Blue</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter Unit, Vial Vent 0.22 &#956;m</td>
			<td>Millec</td>
			<td>SLGP033RS</td>
			<td>Filter used to filter the ashes + 4% HCl Solution</td>
		</tr>
		<tr>
			<td>Scintillation Vials</td>
			<td>Fisherbrand</td>
			<td>03-337-4</td>
			<td>Used to keep the dry powdered plant material during the burning procedure.</td>
		</tr>
		<tr>
			<td>Disposable Syringe 10 ml</td>
			<td>Medi-Plus</td>
			<td>1931</td>
			<td>Syringe used during the filtration</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>231-595-7</td>
			<td>Used in the 4% HCl solution to dilute the ashes and clean the materials</td>
		</tr>
		<tr>
			<td>Tobacco, Nicotiana tabacum cv. Samsun NN</td>
			<td>Obtained from Prof. Simon Barak and routinely used in the Zaccai Lab</td>
			<td>Barak S, Nejidat A, Heimer Y, Volokita M. Transcriptional and posttranscriptional regulation of the glycolate oxidase gene in tobacco seedlings. Plant Molecular Biology. 2001 Mar 1;45(4):399-407.</td>
			<td>Tobacco cultivar used in this protocol</td>
		</tr>
		<tr>
			<td>Glass Wool (Rock Wool)</td>
			<td>Sigma-Aldrich</td>
			<td>659997-17-3</td>
			<td>Used in the procedure of burning samples in the furnace.</td>
		</tr>
	</tbody>
</table>

a colorimetric method for measuring iron content in plants

铁是生物体中最重要的微量营养素之一, 涉及呼吸和光合作用等基本过程。在所有生物体中, 铁含量相当低, 相当于植物的约0.009% 的干重。迄今为止, 测定植物组织中铁浓度的最准确方法之一是火焰吸收原子吸收光谱法。然而, 这种方法耗时且昂贵, 需要在工厂实验室中不常见的特定设备。因此, 需要一个简单而准确的方法, 可以经常使用。在动物和植物组织学切片中, 经常使用比色普鲁士蓝法进行定性铁染色。本研究采用普鲁士蓝法对烟叶中铁进行定量测定。我们验证了这种方法的准确性, 利用原子吸收光谱和普鲁士蓝染色测量相同样品中的铁含量, 并发现两个程序之间的线性回归 (R2 = 0.988)。我们的结论是, 普鲁士蓝法的定量铁测量在植物组织是准确的, 简单的, 廉价的。然而, 在这里提出的线性回归可能不适合其他植物物种, 由于潜在的相互作用的样品和试剂。因此, 需要为不同的植物物种建立一个回归曲线。

当该协议正确执行时, 应在普鲁士蓝和原子光谱学方法取得的结果之间取得良好的相关性。因此, 普鲁士蓝法可以很容易地用于获得准确测量的铁浓度在植物样品, 如以下实验所示。
烟草植物生长如议定书所述, 并灌溉水含有不同的铁浓度 (0, 1, 2, 3, 4, 5, 或6毫米) 超过7天。这些植物在80摄氏度时被收割、清洁和晒干3天。按照所述...

Watch this Scientific Journal Video about A Colorimetric Method for Measuring Iron Content in Plants at JoVE.com

A Colorimetric Method for Measuring Iron Content in Plants

测定植物铁含量的比色法

我们提出了一个简单可靠的协议, 以测量植物组织中的铁含量使用比色普鲁士蓝法。

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

a-colorimetric-method-for-measuring-iron-content-in-plants

Research

JoVE Journal

Biochemistry

21.9K 次观看.  Ben-Gurion University of the Negev. 我们提出了一个简单可靠的协议, 以测量植物组织中的铁含量使用比色普鲁士蓝法。

视频: A Colorimetric Method for Measuring Iron Content in Plants

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

一种测量方法RNA<em&gt;ñ</em<sup&gt; 6</sup&gt; -methyladenosine细胞和组织修改

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

科研

生物化学

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

多聚-PAGE:生物样品中的用于拍摄方法和解决蛋白复合体

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Determination of the Glycogen Content in Cyanobacteria

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have highlighted complex mechanisms of glycogen metabolism, including the diel cycle of biosynthesis and catabolism, redox regulation, and the involvement of non-coding RNA. At the same time, efforts are being made to redirect carbon from glycogen to desirable products in genetically engineered cyanobacteria to enhance product yields. Several methods are used to determine the glycogen contents in cyanobacteria, with variable accuracies and technical complexities. Here, we provide a detailed protocol for the reliable determination of the glycogen content in cyanobacteria that can be performed in a standard life science laboratory. The protocol entails the selective precipitation of glycogen from the cell lysate and the enzymatic depolymerization of glycogen to generate glucose monomers, which are detected by a glucose oxidase-peroxidase (GOD-POD) enzyme coupled assay. The method has been applied to Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002, two model cyanobacterial species that are widely used in metabolic engineering. Moreover, the method successfully showed differences in the glycogen contents between the wildtype and mutants defective in regulatory elements or glycogen biosynthetic genes.

Here, we present a reliable and easy assay to measure the glycogen content in cyanobacterial cells. The procedure entails precipitation, selectable depolymerization, and the detection of glucose residues. This method is suitable for both wildtype and genetically engineered strains and can facilitate the metabolic engineering of cyanobacteria.

测定蓝细菌中的糖原含量

Cyanobacteria accumulate glycogen as a major intracellular carbon and energy storage&#160;during photosynthesis. Recent developments in research have ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

分光光度法测定蓝藻Synechocystis中的蛋白含量

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Using High Content Imaging to Quantify Target Engagement in Adherent Cells

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe validation. Methods that measure this phenomenon without modification of the protein target or small molecule are particularly valuable though technically challenging. The cellular thermal shift assay (CETSA) is one technique to monitor target engagement in living cells. Here, we describe an adaptation of the original CETSA protocol, which allows for high throughput measurements while retaining subcellular localization at the single cell level. We believe this protocol offers important advances to the application of CETSA for in-depth characterization of compound-target interaction, especially in heterogeneous populations of cells.

Measurements of drug target engagement are central to effective drug development and chemical probe validation. Here, we detail a protocol for measuring drug-target engagement using high content imaging in a microplate-compatible adaption of the cellular thermal shift assay (CETSA).

利用高含量成像对增强细胞中的目标参与进行量化

Quantitating the interaction of small molecules with their intended protein target is critical for drug development, target validation and chemical probe ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.