Name: two-dimensional visualization and quantification of labile, inorganic plant nutrients and contaminants in soil
Uploaded: 2020-09-01T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil at JoVE.com

这项研究由奥地利科学基金（FWF）:P30085-N28（托马斯·普罗哈斯卡）和奥地利科学基金（FWF）以及下奥地利联邦州共同资助:P27571-BBL（雅科布·桑特纳）。

作者没有什么可透露的。

此处介绍的溶质成像方案是一种多功能方法，可可视化和量化土壤植物环境中的二维养分和污染物通量。它具有独特的能力，能够产生亚毫米比例的多元素图像的腹腔溶质物种在土壤根界面，超过可实现的空间分辨率的替代方法测量溶质梯度在rhizosphere基本上4。DGT 的定点现场采样方法，结合 LA-ICP-MS 等高度敏感的化学分析方法，有助于详细研究土壤或类似基质中生长的单个植物根部周围的溶质通量动力学。由于基于水槽的取样过程，获得的图像反映了可视化溶解物的易感性，因此估计其植物可用性为10。虽然溶质通量的方法固有的测量具有相当大的优势，如可解释为植物可用的营养成分，但通量测量远不如孔水浓度测量更直接理解。散装土壤应用中的标准 DGT 采样几何形状（特别是该设置中使用的 0.8 毫米厚扩散凝胶）允许通过批量 DGT 测量cDGT和解释这些参数对溶质物种的再补给动力学进行比较实际孔隙水浓度、csoln和时间平均孔水浓度估计值。但是，这种比较不能基于具有非常薄扩散层的成像 DGT 应用程序进行，因为派生的cDGT值是不切实际的小34。因此，DGT成像结果并不总是简单和快速的解释，往往不能直接与更传统的孔隙水浓度测量相提并论。
在应用该方法时，需要仔细考虑一些关键步骤，主要涉及灌装和浇注 rhizotron 生长容器。在将土壤填入 rhizotron 时，避免过多压实土壤非常重要，因为植物根不能穿透强压实的土壤，根的生长将受到抑制。我们观察到根部避免强烈压实的土壤，沿着rhistron生长容器的内缘生长，那里的土壤通常不太紧凑。在这种情况下，位于rhigzotrons中心的单个根，其中DGT凝胶可以方便地应用，可能根本不开发，有效地抑制了成功的凝胶应用。在我们的实验室中，经验表明，1.0-1.4 gcm-3 的干燥土壤散装密度允许不受阻碍的根部发育。此外，过度的土壤压实也是有关氧化物敏感元素和生物地球化学相关物种溶解性的潜在人工来源。由于毛孔总体积减少，毛孔直径分布向高度压实土壤中的低直径转移，因此可用的气填充量较小，从而可能导致局部的还原条件。因此，MnIII/IV- 和 FeIII- 氧化物可能会减少，导致 Mn2+ 和 Fe2+ 通量增加。Fe-oxides的溶解是磷酸盐和微量营养素的重要吸附点，可以解放冰山和/或共同沉淀的物种，从而导致生物地球化学相关物种的人工增高通量。如果生长容器浇水过多，也可能会出现类似的问题。通过生长容器顶部的小土壤表面积蒸发量较低，土壤在种植后可能保持水饱和长达几个星期，这也可能导致重氧化人工制品。
另一个重要考虑因素是制造 HR-DGT 结合凝胶的化学功能。通过遵循协议，获得具有均匀分布的绑定相的薄凝胶。如果凝胶具有不均匀的材料分布区域（例如凝胶或结合相聚合的孔），则需要去除这些区域，或者，如果范围太广，则需要重复凝胶制造协议。如果准备正确，凝胶必须能够结合目标溶胶物种，立即和定量地扩散到凝胶27，这是由分析特异性凝胶结合能力决定的。虽然在未受污染的土壤中，超过凝胶容量的问题较小，但在金属污染土壤和盐碱土壤环境中应予以考虑。凝胶结合相的饱和不仅会损害定量溶胶采样，而且还会导致凝胶中结合相之间的溶解物横向扩散，导致小规模溶胶通量特征的无限期本地化。因此，如果目标土壤环境中预期有大量阴唇养分/污染物物种，则应进行初步测试。对于估计预期的DGT负荷，散装土壤DGT活塞采样，其次是凝胶elution和湿化学分析可以应用15，49。如有必要，可调整 DGT 部署时间以减少凝胶接触时间，从而避免凝胶饱和度超过容量阈值。相反，初步测试也有助于确定所需的凝胶接触时间和/或LA-ICP-MS灵敏度，如果非常低的溶质负荷预期，这可能是重要的映射微量元素溶解物在自然土壤背景水平15。此外，在制定DGT LA-ICP-MS校准标准时，应通过对凝胶的可控加载进行实验应用前验证正确的DGT凝胶功能。凝胶标准提供矩阵匹配的参考凝胶分析加载，可用于评估 LA-ICP-MS 确定的样品凝胶装载是否在预期范围内。如果无法获得与气体和方法空白背景噪声不同的信号，操作员必须确保实施跟踪元素分析的实验室程序，并正确执行所有协议步骤。有时，DGT 凝胶在用土壤暴露的、加载的侧面朝向玻璃板而不是激光束进行溶胶采样后意外翻转，导致最终溶胶通量图像中信号强度低且错误地翻转特征。
在洛杉矶-ICP-MS分析期间，生成了大量数据，这需要相当长的时间进行评估。在我们的实验室中，我们使用针对目标数据输出格式定制的内部数据评估脚本，使用标准电子表格软件。半自动排序和校准后，使用开源、开放访问图像分析工具（ImageJ，斐济50）进行图像绘图。这种方法允许完全控制数据排序、评估和演示，这一点至关重要，因为收集的数据对应于矩形，而不是二次像素，这需要正确地显示在生成的 solute 地图中。此外，在数据处理过程中，应小心避免任何像素插点。像素插点导致化学图像的梯度平滑，导致软化，通常是圆形元素分布特征，因此是原始数据的不良变化。像素插管是许多图像处理软件产品重新缩放和重新格式化操作的标准程序，但通常可以取消选择。
总之，上述方法是了解天然土壤-里佐圈-植物系统中的养分和污染物动力学的重大进展。除了 DGT 专用应用外，该方法还可以与其他基于扩散的成像技术（如平面光学 3、33、42、43、48、51 和 zym 光谱20、21、22、23、24）相结合，并可进一步开发以包括附加元素和土壤参数。

作物植物的营养获取是决定作物产量的关键因素。对作物有效吸收养分的过程进行了深入的研究，特别是通过土壤根系（rhizosphere）的植物根控制养分供应和养分内化的机制，因其在作物养分获取中的作用而得到认可。植物养分吸收的重要过程包括:向根部输送营养:溶解在土壤孔隙水中的物种与与固体土壤表面相连的物种之间的动态吸附平衡:微生物对营养物质的竞争;土壤有机物中所含养分的微生物矿化:和营养内化到根部症状。无机微量金属（oid）污染物的吸收在很大程度上由相同的机制控制。
根据养分和污染物的供应、植物需求和土壤中的扩散性，可以观察到岩石圈中的差异养分模式。对于内化率较高的强吸附元素（例如， P，Fe，Mn，Zn，As，Cd，Pb），与散装土壤相比，阴唇（即可逆吸附）元素部分的耗竭，耗竭区宽度通常≤1毫米，而对于更多的移动养分，如NO3-，耗竭区可以延伸至几厘米1。此外，当可用性超过工厂吸收率2、3时，还观察到 Al 和 Cd 等元素的积累。
鉴于 rhizosphere 过程在养分和污染物循环中的重要性，已开发出几种以高空间分辨率测量植物可用元素分数的技术。然而，测量小规模的腹腔溶胶分布已被证明是具有挑战性的，有几个原因。一个主要困难是在与活植物根相邻的固定位置对非常小（低 μL 范围）的土壤和/或孔水进行采样，以解决 rhizosphere 中陡峭的养分梯度。解决这个问题的方法之一是使用微吸杯提取孔隙水样品6。通过这种方法，A. Güttlein、A. Heim 和 E. Matzner7测量了奎尔库斯罗布尔L. 根附近的土壤孔水养分浓度，其空间分辨率为 +1 厘米。分析 μL 土壤或土壤溶液量的一个困难是，这些小样本量与除主要养分物种外的所有低浓度相结合，需要高度敏感的化学分析技术。
另一种系统，能够解决营养梯度在分辨率下降到〜0.5毫米，是种植根垫在土壤块的表面，与薄亲水膜层分离土壤从根8，9。在这种配置中，溶胶可以穿过膜，根可以从土壤中产生养分和污染物，而根渗出物可以扩散到土壤中。建立密集的根层后，可以对土壤块进行取样和切片，以获得土壤样本，以便随后提取元素部分。通过这种方式，可以分析在相对较大的区域（约100厘米2）平均的一维营养素和污染物梯度。
另一个挑战是获得实验室、植物可用元素部分的样本，因为大多数化学土壤提取技术的运作方式与植物获取养分和污染物的机制大相径庭。在许多土壤提取协议中，土壤与萃取溶液混合，目的是在溶解元素和冰激元素部分之间建立（伪）平衡。然而，植物不断内化养分，因此，往往逐渐耗尽了里佐圈的土壤。虽然均衡提取方案已被广泛采用作为土壤测试，因为它们很容易实施，提取的营养成分往往不代表植物可用的营养成分井10，11，12，13。不断耗尽被采样土壤养分的水槽方法已被建议为有利的方法，并可能更类似于潜在的养分吸收机制，模仿根吸收过程10，11，14，15。
除了上述方法外，还开发了能够测量分辨率为≤100μm的连续参数图的真人成像应用，用于特定元素和土壤（生物）化学参数5。自传可以用来成像元素分布在日光圈，只要有合适的放射性同位素16。平面光电图可可视化重要的土壤化学参数，如pH值和pO217、18、19，酶活性或蛋白质总分布可以使用荧光指示器成像技术（如土壤成像20、21、22、23和/或根部印迹方法24）进行映射。虽然酶成像和自传仅限于一次测量单个参数，但使用平面光电图进行 pH 和 pO2成像可以同时完成。更传统的根垫技术仅提供一维信息，而微吸盘提供点测量或低分辨率二维信息，但两种方法都允许多元素分析。最近，P.D. Ilhardt等人提出了一种利用激光诱导分解光谱（LIBS）绘制2D总多元素分布图的新方法，分辨率为+100μm的土壤根芯样品，通过仔细的样品准备保留了自然元素分布。
唯一能够以高空间分辨率对多种营养物质和污染物溶解物进行二维取样的技术是薄膜（DGT）技术中的扩散梯度，这是一种基于水槽的取样方法，可在嵌入在水凝胶层26、27层的结合材料上原位固定可移动的实验室微量金属（loid）物种。DGT作为测量沉积物和水域中腹腔溶解物的化学规格技术被引入，并很快被用于土壤28。它使亚毫米比例的多元素溶胶成像，这是最初在河流沉积物29中演示，并已进一步开发，以应用于植物里佐圈30，31，32，33。
对于 DGT 取样，在土壤块表面层生长的单个植物根上涂上一块大小约为 3 厘米 x 5 厘米的凝胶片，用亲水膜将凝胶与土壤分离。在接触期间，阴唇营养物质和/或污染物扩散到凝胶上，并立即被凝胶中加入的结合材料所束缚。通过这种方式，在采样过程中建立了浓度梯度，从而建立了对凝胶的连续净通量。取样后，可以使用分析化学技术去除和分析水凝胶，从而进行空间解析分析。为此目的，一项高度专业化且常用的技术是激光消融感应耦合等离子体质谱仪 （LA-ICP-MS）。在一些早期研究中，微粒子诱导的X射线发射（PIXE）也被使用29。DGT 采样与 LA-ICP-MS 分析相结合，可实现空间分辨率为 100μm 的多元素化学成像。如果采用高度敏感的 ICP-MS 技术（例如部门领域 ICP-MS），则可以达到极低的检测限制。在一项有关玉米15号对Zn和Cd吸收的影响的研究中，我们能够绘制出未受污染土壤中玉米里佐圈的阴唇CD，每个凝胶面积的检测限量为38 pgcm-2。DGT、平面光学和酶学依赖于目标元素从土壤扩散到凝胶层，这些凝胶层可用于这些方法的联合应用，以便同时或连续地成像与植物养分和污染物吸收相关的大量参数。关于DGT成像分析化学方面的详细信息，关于DGT和其他成像方法相结合的潜力，以及它的应用，在参考第34，35中进行了全面审查。
本文介绍了如何利用DGT技术对不饱和土壤环境中的陆生植物根部进行溶解成像实验，包括植物栽培、凝胶制造、凝胶应用、凝胶分析和图像生成。所有步骤都详细阐述，包括关于关键步骤和实验备选方案的说明。

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;8&lt;/sub&gt; (ammonium persulfate; APS)</td><td>VWR</td><td>21300.260</td><td>&amp;ge;98.0%, analytical reagent</td></tr><tr><td>2-(N-morpholino)-ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>M8250-100G</td><td>&amp;ge;99.5%</td></tr><tr><td>Acrylamide solution</td><td>Sigma-Aldrich</td><td>A4058-100ML</td><td>40%, for electrophoresis</td></tr><tr><td>Analyte salts</td><td>n/a</td><td>n/a</td><td>Use water soluble analyte salts of analytical grade or higher</td></tr><tr><td>Buechner funnel</td><td>VWR</td><td>511-0065</td><td>13 cm plate diameter</td></tr><tr><td>Chemical equilibrium modelling software</td><td>KTH Sweden</td><td>n/a</td><td>Visual MINTEQ</td></tr><tr><td>Clamp</td><td>Local warehouse</td><td>n/a</td><td></td></tr><tr><td>Desktop publishing software</td><td>Adobe Inc.</td><td>n/a</td><td>InDesign CS6</td></tr><tr><td>DGT cross-linker</td><td>DGT Research Ltd</td><td>n/a</td><td>2%, agarose derivative</td></tr><tr><td>DGT piston sampler</td><td>DGT Research Ltd</td><td>n/a</td><td>2 cm diameter exposure window</td></tr><tr><td>Digital single-lens reflex (DSLR) camera</td><td>Canon Inc.</td><td>n/a</td><td>Canon EOS 1000D</td></tr><tr><td>Dispersion device</td><td>IKA</td><td>3737000</td><td>Ultra-Turrax T10 Basic</td></tr><tr><td>Double-sided adhesive tape</td><td>Tesa</td><td>56171</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>34923</td><td>Puriss. p.a., absolute, &amp;ge;99.8%</td></tr><tr><td>Gel blotting paper</td><td>Whatman</td><td>10426981</td><td>Blotting Papers, Grade GB005, 20 &amp;times; 20 cm, 1.5 mm thickness</td></tr><tr><td>Gel drier</td><td>UniEquip</td><td>n/a</td><td>UNIGELDRYER 3545</td></tr><tr><td>High-pressure microwave system</td><td>Anton Paar</td><td>n/a</td><td>Multiwave 3000</td></tr><tr><td>HNO&lt;sub&gt;3&lt;/sub&gt;</td><td>VWR</td><td>1.00456.2500P</td><td>65%, ISO for analysis</td></tr><tr><td>Horizontal shaker</td><td>GFL</td><td>305</td><td></td></tr><tr><td>HydroMed D4</td><td>AdvanSource Biomaterials Corp.</td><td>n/a</td><td>Ether-based hydrophilic urethane</td></tr><tr><td>ICP-MS software</td><td>Perkin Elmer</td><td>n/a</td><td>Syngistix</td></tr><tr><td>Image analysis software</td><td>National Institutes of Health (NIH)</td><td>n/a</td><td>ImageJ Fiji, freely available at https://fiji.sc/</td></tr><tr><td>Knife-coating device</td><td>BYK</td><td>5561</td><td>Single Bar 6&amp;Prime;, 0.5 mils</td></tr><tr><td>LA software</td><td>Elemental Scientific Lasers</td><td>n/a</td><td>ActiveView</td></tr><tr><td>LA system</td><td>Elemental Scientific Lasers</td><td>n/a</td><td>NWR193</td></tr><tr><td>Laminar flow bench</td><td>Telstar Laboratory Equipment B.V.</td><td>n/a</td><td>Class II biological safety cabinet</td></tr><tr><td>Magnetic stirrer</td><td>IKA</td><td>0003582400</td><td>C-MAG MS 7</td></tr><tr><td>Moisture-retaining film</td><td>Bemis Company, Inc.</td><td>PM999</td><td>Parafilm M, 4&quot; x 250&#039;</td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich</td><td>T9281-50ML</td><td>BioReagent, suitable for electrophoresis, ~99%</td></tr><tr><td>NaNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>229938-10G</td><td>99.995% trace metals basis</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>1064980500</td><td>Pellets for analysis</td></tr><tr><td>Overhead shaker</td><td>GFL</td><td>3040</td><td></td></tr><tr><td>Perfluoroalkoxy alkane (PFA) vials</td><td>Savillex</td><td>200-015-20</td><td>15 mL Standard Vial, Rounded Interior</td></tr><tr><td>pH meter</td><td>Thermo Scientific</td><td>13-644-928</td><td>Orion 3-Star Benchtop pH Meter</td></tr><tr><td>pH probe</td><td>Thermo Scientific</td><td>8157BNUMD</td><td>Orion ROSS Ultra pH/ATC Triode</td></tr><tr><td>Plastic cutter</td><td>DGT Research Ltd</td><td>n/a</td><td>Use empty cross-linker vials from DGT research Ltd</td></tr><tr><td>Plastic tweezers</td><td>Semadeni</td><td>602</td><td></td></tr><tr><td>Plasticine</td><td>Local stationary shop</td><td>n/a</td><td>non-drying plastic modelling mass based on paraffin wax and bulking agents</td></tr><tr><td>Polycarbonate membrane discs</td><td>Whatman</td><td>110606</td><td>Nuclepore Hydrophilic Membrane, 25 mm diameter, 0.2 &amp;micro;m pore size, 10 &amp;micro;m thickness</td></tr><tr><td>Polycarbonate membrane sheet</td><td>Whatman</td><td>113506</td><td>Nuclepore Hydrophilic Membrane, 8 &amp;times; 10 in, 0.2 &amp;micro;m pore size, 10 &amp;micro;m thickness</td></tr><tr><td>Polyethersulfone membrane discs</td><td>Pall Corporation</td><td>60172</td><td>Supor 450 Membrane Disc Filters, 25 mm diameter, 0.45 &amp;micro;m pore size, 0.14 mm thickness</td></tr><tr><td>Polyethersulfone membrane sheet</td><td>Pall Corporation</td><td>60179</td><td>Supor 450 Membrane Disc Filters, 293 mm diameter, 0.45 &amp;micro;m pore size, 0.14 mm thickness</td></tr><tr><td>PTFE foil</td><td>Haberkorn</td><td>n/a</td><td>50 &amp;micro;m thickness</td></tr><tr><td>PTFE spacer</td><td>Haberkorn</td><td>n/a</td><td>Variable thicknesses available</td></tr><tr><td>PTFE-coated razor blades</td><td>Personna GEM</td><td>62-0178</td><td>Stainless steel single edge blades (coated)</td></tr><tr><td>PTFE-coated Tygon tubing</td><td>S-prep GmbH</td><td>SP8180</td><td>0.32 cm inner diameter</td></tr><tr><td>Quadrupole ICP-MS</td><td>Perkin Elmer</td><td>N8150044</td><td>NexION 2000B</td></tr><tr><td>Quantitative filter paper, 454</td><td>VWR</td><td>516-0854</td><td>Particle retention 12-15 &amp;micro;m</td></tr><tr><td>Spreadsheet software</td><td>Microsoft Corporation</td><td>n/a</td><td>Microsoft Excel 2016 (v16.0)</td></tr><tr><td>Stainless-steel cutter</td><td>Local locksmithery</td><td>n/a</td><td>2.5 cm diameter</td></tr><tr><td>Suspended particulate reagent-iminodiacetate (SPR-IDA)</td><td>Teledyne CETAC Technologies</td><td>n/a</td><td>10 &amp;micro;m diameter polystyrene beads, 10 % (w/v) bead suspension</td></tr><tr><td>Transistor-transistor logic (TTL) cable</td><td>n/a</td><td>n/a</td><td>Consult ICP-MS technician to identify a suitable TTL cable for a specific instrument</td></tr><tr><td>Two-volume cell</td><td>Elemental Scientific Lasers</td><td>n/a</td><td>Two-volume cell 1</td></tr><tr><td>Vinyl electrical tape</td><td>3M</td><td>n/a</td><td>Scotch Super 33+</td></tr><tr><td>Water purification system</td><td>Termo Electron LED GmbH</td><td>n/a</td><td>TKA-GenPure</td></tr><tr><td>ZrOCl&lt;sub&gt;2&lt;/sub&gt; &amp;times; 8H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Alfa Aesar</td><td>86108.30</td><td>99.9 %, metals basis</td></tr></tbody></table>

two-dimensional visualization and quantification of labile, inorganic plant nutrients and contaminants in soil

我们描述了一种二维（2D）可视化和量化阴唇分布的方法（即 逆向吸收）无机养分（如P、Fe、Mn）和污染物（如Cd、Pb）以低于毫米（约100微米）的空间分辨率在植物根部（"rhizosphere"）相邻的土壤中溶解物种。该方法结合了薄膜（DGT）技术中扩散梯度的沉基溶胶采样与激光消融感应耦合等离子质谱（LA-ICP-MS）的空间解化学分析。DGT 技术基于具有均匀分布分析器选择性结合相的薄水凝胶。可用绑定阶段的多样性允许在简单的凝胶制造程序后制备不同的 DGT 凝胶类型。对于 Rhizosphere 中的 DGT 凝胶部署，植物生长在平坦、透明的生长容器 （rhizotrons） 中，从而能够以最小的侵入性进入土壤生长的根系。在生长前阶段后，DGT 凝胶应用于某些感兴趣的区域，用于在 rhizospher 中进行原位溶胶采样。之后，使用 LA-ICP-MS 线扫描成像检索并准备随后对绑定溶胶进行化学分析。使用 13C 应用内部规范化，使用矩阵匹配凝胶标准进行外部校准，进一步允许 2D 溶质通量的量化。该方法在土壤植物环境中生成多元素溶质通量的定量、亚毫米比例二维图像的能力方面独树一帜，大大超过了测量日光圈中溶质梯度的其他方法的可实现空间分辨率。我们介绍了在陆地植物的岩石圈中成像多成离子和肛门溶质物种的方法的应用和评价，并强调了将这种方法与补充溶质成像技术相结合的可能性。

为了证明DGT成像方法的能力和数据细节，我们汇编了与法戈皮鲁姆树脂和萨利克斯·史密西亚纳（图7）相邻的土壤中多种阴唇营养素和污染物溶质物种的亚毫米、二维通量分布。协议的一般程序步骤的大致时间在表1中。
图 7中的索卢特图像是在三种不同的研究中使用 HR-MBG 或 HR-CBG 绑定凝胶生成的。化学图像显示沿 x 轴的空间分辨率为 82-120 μm，沿 y 轴的 300-400 μm 的 2D 溶质通量分布，具体取决于所使用的 LA-ICP-MS 参数。由于在图像校准和调整过程中没有应用插点，因此单个像素表示已测量的数据点。将溶胶图像与 ROI 的摄影图像对齐后发现，根据土壤结构和根部形态，不同元素的亚毫米、二维溶质通量分布高度可变。这可以归因于土壤-红球-植物系统中元素的微分生化行为，以及它们与土壤矩阵和植物根的相互作用。
在 图7A 阴唇无机Mg，Al，P，Mn和Fe溶胶通量被可视化周围的一个年轻的 F.埃斯库伦特 根生长在无碳酸盐土壤施肥与NH4NO3.亚毫米溶质分布显示，由于根吸收，Al、P 和 Fe 通量与旧根部分一起减少，由于 F. esculentum 根的局部 P 动员过程，根部顶点的 Mg、Al、P、Mn 和 Fe 通量高度增加。请注意，根尖位于土壤表面后面，因此在照片图像中几乎看不到。 图 7B 显示了在受 Zn、Cd 和 Pb 适度污染的土壤中生长的耐金属 S. Smithiana 根部周围的阴唇微量金属的分布，包括 Mn、Fe、Zn、Cd 和 Pb。溶胶图像可视化了明显的消耗，特别是 Zn、Cd 和 Pb 的直接根部，表明 S. Smithiana 根部充当受污染土壤中腹腔微量金属的局部沉降器。此外，还可以观察到局部 Zn、Cd 和 Pb 通量增加，表明这些微量金属在直接土壤根接口的积累。
除了多元素溶胶成像外，该方法还可以与基于增释的辅助成像技术（如平面光学34）相结合。这一点在图7C中得到了证明，其中在S.史密斯尼亚纳的rhizospher中，阴唇微量金属的分布与pH值的分布是通过结合的单层平面光电-DGT cation结合凝胶33共同本地化的。土壤基板用 （NH4）2SO4施肥，导致与散装土壤相比，根轴沿线的 pH 降低 1 单位。pH 的下降与 Mn、Fe、Co、Ni、Cu 和 Pb 的溶质通量增加共同本地化，表明 pH 引起的金属溶解。
此外，这些示例结果显示了可能获得的一些潜在的成像人工制品。例如，结构土壤的不均匀性，例如，在 图 7A的 ROI 图像的下三分之一中观察到水平线，可导致土壤凝胶接触不连续性，导致此位置的有限扩散到粘合凝胶中。相反，在rhiozotron中过度的土壤压实会导致孔隙性差，导致土壤重氧化状态向厌氧的人工转移。 图 7B中说明了这一点，其中溶质图像中高度升高的 Mn 和 Fe 通量的大面积区域与 ROI 图像中的密集土壤层在视觉上相匹配。这表明，由于土壤压实，土壤重氧化的可能性降低，导致高度重氧化敏感元素的还原溶解和溶解。因此，建议在灌装后直接对土壤表面进行仔细的 rhizotron 填充和目视检查。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61661/61661fig07.jpg"> 图7:不同土壤根界面的阴唇养分和污染物溶质物种的亚毫米二维分布。（A） 在年轻的F. 埃斯库伦图姆根周围分布的食人性 P 和 cationic Mg、Al、Mn 和 Fe 溶质。在=75%WHC的土壤水饱和度下，使用HR-MBG对阴离子和阴离子溶胶进行了24小时的联合局部取样。Al、P 和 Mn 图像显示为校准fDGT值（pg cm-2 s-1），而 Mg 和 Fe 图像显示13C 规范化强度。比例尺条代表 1 厘米。这个数字是根据参考48改编的。（B） 在中度受Zn、Cd和Pb污染的土壤中生长的S.smithiana根周围分布Mn、Fe、Zn、Cd和Pb。所有图像都显示为校准的 fDGT值 （pg cm-2 s-1）。刻度条代表 0.5 厘米。这个数字是根据参考3改编的。（C） 通过修改HR-MBG协议，实现了在土壤中生长的S.Smithiana根部周围的pH和多个cationiana溶解物的分布，并采用CD.pH和溶胶动力学的联合定位，允许同时进行溶胶采样和平面光电成像33。 Mn、Cu、Zn 和 Cd 图像以校准fDGT值（pg cm-2 s-1）显示，而 Fe、Co、Ni 和 Pb 图像显示13个 C 规范化强度。比例尺条代表 1 厘米。这个数字是根据参考33改编的。所列数字转载自引用的第3条、第33条、第48条，这些条文根据CC BY获得许可。<a href="https://www.jove.com/files/ftp_upload/61661/61661fig07large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
补充文件 1.<a href="https://www.jove.com/files/ftp_upload/61661/JoVE61661_Supporting%20Information_Updated%202021_.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil at JoVE.com

Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

此协议提供了多个阴唇无机养分和污染物溶胶物种的亚毫米 2D 可视化工作流程，使用薄膜 （DGT） 中的扩散梯度与质谱成像相结合。详细描述了在陆地植物的岩石圈中对溶解物进行定量测绘的索卢特采样和高分辨率化学分析。

土壤中实验室、无机植物养分和污染物的二维可视化和量化

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

two-dimensional-visualization-quantification-labile-inorganic-plant

Nanyang Technological University

Research

JoVE Journal

Environment

6.0K 次观看.  Montanuniversität Leoben. 此协议提供了多个阴唇无机养分和污染物溶胶物种的亚毫米 2D 可视化工作流程，使用薄膜 （DGT） 中的扩散梯度与质谱成像相结合。详细描述了在陆地植物的岩石圈中对溶解物进行定量测绘的索卢特采样和高分辨率化学分析。

视频: Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

综合外地Lysimetry和孔隙水采样的化学活动性土壤的评价和植被成立

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

科研

环境科学

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.

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.

植物与放射性示踪剂测量矿质营养和毒物的通量

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

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

土壤蒸渗仪开挖加之水文，地球化学和微生物调查

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

活性有机碳评估土壤中连续熏蒸孵化程序

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

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.

土壤联合粒径与密度的分离研究

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

Measurements of Soil Carbon by Neutron-Gamma Analysis in Static and Scanning Modes

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of gamma rays created when neutrons interact with soil elements. The main parts of the INS system are a pulsed neutron generator, NaI(Tl) gamma detectors, split electronics to separate gamma spectra due to INS and thermo-neutron capture (TNC) processes, and software for gamma spectra acquisition and data processing. This method has several advantages over other methods in that it is a non-destructive in situ method that measures the average carbon content in large soil volumes, is negligibly impacted by local sharp changes in soil carbon, and can be used in stationary or scanning modes. The result of the INS method is the carbon content from a site with a footprint of ~2.5 - 3 m2 in the stationary regime, or the average carbon content of the traversed area in the scanning regime. The measurement range of the current INS system is &#62;1.5 carbon weight % (standard deviation &#177; 0.3 w%) in the upper 10 cm soil layer for a 1 hmeasurement.

Here, we present the protocol for in situ measurement of soil carbon using the neutron-gamma technique for single point measurements (static mode) or field averages (scanning mode). We also describe system construction and elaborate data treatment procedures.

静态和扫描模式下中子伽马分析法测量土壤碳

The herein described application of the inelastic neutron scattering (INS) method for soil carbon analysis is based on the registration and analysis of ...

工程学

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

监测农业土壤中经修正的硅酸盐矿物风化造成的致病性无机碳积累。

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

元素成像保护湿地根和岩石圈的方法

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...