作者感谢欧盟 H2020 计划（项目 GAIN4CROPS，GA 编号 862087）。AgroCropFuture 农业生态学和未来气候中的新作物卓越中心由爱沙尼亚教育和研究部资助。我们要感谢 Evelin Loit-Harro 教授提供 T. aestivum 和 Z. mays 的种子，感谢 Paula Palmet 和 Vaiko Vainola 在准备叶横截面方面的帮助，以及 João Paulo de Silva Souza 在分析方面的帮助。所有图像均来自爱沙尼亚生命科学大学的显微镜部门，在各种项目下。

1. 植物生长条件
<ol><li>分别选择小麦 （Triticum aestivum L. Var. 冬小麦， "Fredis"） 和玉米 （Zea mays L. Var. Saccharata， "Golden Bantam"） 作为代表性的 C3 和 C4 植物。在环境受控的生长室中用种子培养这两种植物。</li>
	<li>将相对湿度保持在 55%，空气温度为 25 °C/18 °C（昼/夜），光照时间为 12 小时。将植物表面的光合光子通量密度 （Q） 保持在 1200 μmol/m2/s，以代表其自然生长条件。在 400 μmol/mol 的环境 CO2 浓度下种植所有植物。</li>
	<li>每2 天用 蒸馏水给植物浇水，并在幼苗出苗后 10 天用 Hoagland 溶液施肥。在给定条件下将植物生长 60 天，并使用完全展开的叶子进行显微镜分析。</li>
</ol>2. 手绘部分的准备和查看
<ol><li>从每株植物中选择 3 片成熟的非衰老叶子，并将它们保存在蒸馏水中。使用新的单面剃须刀片，进行一次平整切割，与叶子表面形成直角，以确保截面是垂直于叶子表面的横向横截面。</li>
	<li>将叶标本放在玻璃板上的一块牙蜡上，以稳定样品并避免滑倒。通过在叶标本上笔直向下移动刀片进行切割，以干净地切开细胞。将横截面保存在蒸馏水中。</li>
	<li>将样品放在水滴上并用玻璃盖玻片覆盖，以便显微镜观察和成像，从而将样品安装在载玻片上。避免积聚气泡。</li>
	<li>在复合光学显微镜下以 10 倍和 20 倍放大倍率观察载玻片。根据软件指南保存图像以及相关比例。</li>
</ol>3. 半薄片的制备
<ol><li>如步骤 2.2 所示，在玻璃板上从 Z. mays 和 T. aestivum 的叶子上从中肋区沿肋间区域切下约 3 毫米 x 5 毫米大小的切片。确保切割部分的长度沿着叶子的纹理延伸。</li>
	<li>将植物材料放入装有 1 mL 固定缓冲液（4% 戊二醛在 0.1 M 磷酸盐缓冲液中，pH = 6.9， 表 1）的注射器中。</li>
	<li>垂直握住注射器，去除空气，然后用手指放在尖端，泵送注射器以产生真空。重复直到样品位于注射器底部，表明渗透成功。</li>
	<li>将注射器的内容物转移到标记的玻璃瓶中，并在下一步之前在 4 °C 下储存 48 小时（表 2）。</li>
	<li>对于每个小瓶，用移液管轻轻去除固定剂并加入磷酸盐缓冲液，直到样品被覆盖。静置 30 分钟。重复此步骤 3 次。</li>
	<li>用移液管除去磷酸盐缓冲液，并加入四氧化锇 （CAUTION） 直至样品被覆盖。样品和其他有机物会变黑。使用双层手套。放置 1 小时。</li>
	<li>用移液管除去之前的溶液，并用蒸馏水覆盖样品。放置 30 分钟 重复此步骤 3 次，并戴上双层手套。</li>
	<li>用移液管除去前一种溶液，并用 50/50 蒸馏水-乙醇溶液覆盖样品。静置 30 分钟。使用以下系列的蒸馏水-乙醇溶液重复此步骤:30/70、20/80、10/90 和 2x，100% 乙醇。这些溶液中的任何样品都可以在 4 °C 下放置过夜。</li>
	<li>用移液管除去前一种溶液，并用 1/3 树脂-乙醇溶液覆盖样品。将样品放在搅拌板上，放置 2 小时。使用以下系列的树脂-乙醇溶液重复此步骤:1/2、1/1、2/1、3/1 和 2x，100% 树脂。这些溶液中的任何样品都可以在 4 °C 下放置过夜。</li>
	<li>用 100% 树脂覆盖平面包埋模具的型腔（16 个型腔，型腔尺寸:14 L x 4.8 W x 3 D （mm）），并将样品放在型腔的一端。为每个样品准备铅笔书写的纸质标签，并将它们放在腔体的另一端。</li>
	<li>用 100% 树脂完全填充所有腔体，如果样品和标签移动，请将其拉直。用嵌入膜覆盖模具，并按照丙烯酸树脂产品指南在烘箱中聚合。</li>
	<li>将带有样品的聚合块放在超薄切片机的样品架中。使用粗糙的玻璃刀修剪块，直到组织变得可见并去除多余的树脂。</li>
	<li>使用玻璃或金刚石刀横向 （1 μm） 切割半薄片。使用金属接种环从水面收集切片，并将它们放在载玻片上。在热板 （60 °C） 上干燥载玻片以将切片固定在玻璃上。</li>
	<li>用甲苯胺蓝（1% 水溶液）染色固定切片 5 秒，然后用蒸馏水冲洗并在热板上再次干燥。 注意:确保在通风橱下进行化学固定过程。应定期更换玻璃刀，以确保切割锋利，使切片没有撕裂或变形。</li>
</ol>4. 样品成像
<ol><li>新鲜叶切片的成像<ol><li>根据手册使用随附的软件设置复合光学显微镜。</li>
		<li>将载玻片放在载物台上。调整目镜并以 10 倍观察截面以获得概览，然后在 20 倍和 40 倍物镜下观察。</li>
		<li>在每个目标处，导航 Live 频道，专注于感兴趣的区域，并找到静脉周围的束鞘细胞。</li>
		<li>聚焦后，单击 冻结 按钮并通过按 比例 尺 按钮 工具 选项卡。将图像存储在 中。TIF 格式进行图像分析。</li>
	</ol></li>
	<li>树脂浸润切片的成像<ol><li>将自动光学显微镜与计算机上的随附软件相结合。</li>
		<li>加载透明通道并将载玻片放在显微镜载物台上。使用 40 倍物镜放大倍率聚焦切片并调整亮度以确保所有细胞结构都可见。</li>
		<li>使用导航器功能设置截面的位置区域，以确保整个截面都成像，然后单击 Stitch 按钮。</li>
		<li>单击 Done（完成 ），然后单击 Start（开始 ），以允许显微镜对整个切片进行成像。打开图像分析软件并打开显微镜拍摄的图块。</li>
		<li>使用 Align 功能，确保切片不重叠。生成图像后，使用 调整 标签。</li>
		<li>在存储之前在图像上打印比例尺。TIF 格式。</li>
	</ol></li>
	<li>使用 ImageJ 软件进行解剖测量<ol><li>打开 ImageJ 软件并通过将图像拖到窗口中来加载图像。</li>
		<li>使用"线条"工具，按如下方式校准比例:在比例尺上绘制一条线，选择 "分析">"设置比例"，将已知距离更改为比例尺的长度，然后将长度单位更改为正确的单位。</li>
		<li>通过选择 Line Tool，在感兴趣区域画一条线，然后按 M 键来测量预期特征。</li>
		<li>对于面积测量，请选择 多边形选择工具 并在感兴趣的组织周围绘制。按 M 键结束。测量值显示为弹出窗口，可以将其复制到电子表格中以进行进一步的数据分析。</li>
	</ol></li>
</ol>

提高 C4 作物光合效率的显微镜分析

作者声明没有利益冲突。

在本文中，我们讨论了测量叶片解剖结构的定量和定性方法以及优化它们的方法。此外，该方法应用于代表性作物物种，以确定哪些解剖性状在区分 C3 和 C4 横截面时最有用。了解这些特征至关重要，因为被称为 C2 光合作用的杂交物种正在成为一种更有前途的研究途径。截至目前，只有一种作物物种 Diplotaxis tenuifolia （芝麻菜）被确定使用 C2 光合作用，但?...

光合作用是将光能转化为化学能的基本过程，是陆地营养网络的基石。大多数植物遵循光合作用的 C3 途径，其中主要的光合作用产物是三碳化合物甘油酸酯 3-磷酸盐。C3 光合作用是在 20 多亿年前在大气中进化而来的，其中 CO2 含量丰富，O21 含量较低。在这些条件下进化的关键光合酶核酮糖 1,5 二磷酸羧化酶/加氧酶 （Rubisco） 在当前低 CO2 高 O2 条件下是次优的，因为它与 O2 竞争性反应，启动光呼吸2。光呼吸是一种浪费的途径，它消耗能量而不是产生能量并释放 CO2 作为副产品。因此，在叶绿体中 Rubisco 周围保持高 CO2 浓度以防止氧合至关重要 3,4。由于 C3 植物无法浓缩 CO2，因此 CO2 从环境空气中大量吸收到叶绿体中，抑制光合作用并影响植物生长和生物质生产 2,5,6。
在 C3 植物中，光合作用受到 CO2 通过气孔进入、通过叶肉扩散以及光合酶的生化活性的限制。CO2 的进入首先受到气孔导度的限制，气孔导度受空气温度和湿度等环境条件控制。然后 CO2 通过叶子的内部气相和液相扩散到 Rubisco7。在 C3 植物中，光合作用的所有阶段都发生在叶肉细胞的叶绿体中，植物需要保持 CO2 从大气中持续流入叶绿体。叶绿体中 CO2 的可用性取决于气孔开放性、叶肉结构以及单个细胞和叶绿体特性，这使得植物容易受到环境限制的影响，最终影响光合作用，如低水可用性和高温 7,8,9,10，特别突出了它们对气候变化条件的脆弱性11。
鉴于 C3 途径的低效率带来的挑战，以及维持最佳 CO2 水平和对环境因素的敏感性的限制，在某些植物中，另一种途径，即 C4 光合作用途径，已经进化出来。特征性地，C4 植物具有两条空间上分离的生化途径;磷酸烯醇式丙酮酸羧化酶在叶肉细胞中发生初始 CO2 固定，磷酸烯醇式丙酮酸羧化酶对 CO2 的亲和力高于 Rubisco，并且也缺乏氧合活性。形成的 C4 产物进一步转运到束状鞘细胞，在那里脱羧，CO2 再次被 Rubisco（C3 光合作用）释放和固定12,13,14。PEP 羧化酶对 CO2 的亲和力更大，束鞘细胞的厚细胞壁允许 CO2 在束鞘细胞中浓缩，因此，C4 植物通过空间分离 CO2 固定和卡尔文循环来最大限度地减少光呼吸。C4 途径的采用展示了大自然对环境限制的适应性反应，为在不断变化的气候条件下提高作物生产力和复原力的潜在策略提供了见解15。
C4 植物叶结构的专门解剖结构的特征是叶脉被含有叶绿体的扩大的维管束鞘细胞包围，并且叶肉细胞呈放射状排列，外环围绕束鞘细胞。叶肉细胞靠近束鞘细胞，这使得代谢物能够在两种细胞类型之间快速连续地运输。该细胞的排列是典型的 C4 植物，称为 Kranz 解剖学16。在 C3 物种中，叶肉细胞的特化和处置可能会有所不同，但束鞘细胞明显较小，并且有一些叶绿体或根本没有叶绿体。特异性 Kranz 解剖结构允许将 CO2 集中在 C3-羧化酶 Rubisco 所在的束鞘细胞中的叶绿体中，从而有效阻碍光呼吸 4,17,18。尽管其看似复杂的排列，但这些变化在被子植物的进化中独立地多次发生，表明它是一个相对可行的进化途径 19,20,21，并且各种分类群已被证明处于 C 3 和 C4 碳代谢之间的中间阶段，称为 C3-C 4 或 C 2， 具有浓缩和重新吸收 CO2 22,23,24,25 的能力。
许多 C4 植物是具有高度经济重要性的作物，对 C3 作物（如水稻）进行基因工程改造以提高其气候适应力并确保产量一直是过去几十年来感兴趣的话题26,27。然而，工程工作需要详细了解 C4 特化解剖结构及其如何控制光合作用 2,28。
建立 C4 Kranz 解剖结构是实现将 C4 光合作用工程化为 C3 作物的雄心勃勃目标的先决条件25。然而，目前对 Kranz 解剖学的调节和快速筛选其关键解剖特征的方法的理解是有限的，因此很难识别杂交物种。先前的研究表明，调节 C3 和 C4 植物光合效率的关键性状包括脉间距离、束鞘复合体的直径和束鞘细胞的大小14,29。这些性状可以使用徒手切片轻松筛选，并使用半薄切片进行定量分析。在这里，我们描述了通过徒手交叉和光学显微镜评估允许 C3 和 C4 解剖区分诊断的性状的方法，即束鞘面积、静脉间距离和静脉频率。

<table><tbody><tr><td>Disodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;2H&lt;sub&gt;2&lt;/sub&gt;O) pure</td><td>PENTA, CZ</td><td>10028-24-7</td><td></td></tr><tr><td>Embedding Film, 7.8 mil Thick, 8 x 12.5, (203 x 318mm)</td><td>ACLAR, US</td><td>10501-10</td><td></td></tr><tr><td>Ethanol, abs. 100% a.r.</td><td>Chem-Lab NV, BE</td><td>CL00.0505.1000</td><td>Danger: Highly inflammable liquid and vapour.</td></tr><tr><td>EVOS Invitrogen FL Auto 2 Imaging System</td><td>Thermo Fisher Scientific, US</td><td></td><td></td></tr><tr><td>Flat Embedding PTFE Mold with Metal Frame, 16 cavities</td><td>PELCO, US</td><td>10501</td><td></td></tr><tr><td>Glass vial 2 ml</td><td>VWR Life Science, US</td><td>548-0045</td><td></td></tr><tr><td>Glutaraldehyde 50% solution</td><td>VWR Life Science, US</td><td>23H2856331</td><td>Danger: Fatal if inhaled. Toxic if swallowed. Causes severe skin burns and eye damage. May cause respiratory irritation. Wear protective gloves, protective clothing, eyes and face protection.</td></tr><tr><td>Histo diamond knife</td><td>Diatome, US</td><td></td><td></td></tr><tr><td>LEICA EM UC7</td><td>Leica Vienna, AT</td><td></td><td></td></tr><tr><td>LR White resin hard grade</td><td>Electron Microscopy Sciences, US</td><td>14383</td><td>Danger: Causes skin irritation. Causes severe eye irritation May cause respiratory irritation. May cause drowsiness or dizziness Wear protective gloves, protective clothing, eyes and face protection.</td></tr><tr><td>Microscope slides</td><td>Normax, PT</td><td>5470308A</td><td></td></tr><tr><td>Nikon Eclipse E600 and Nikon DS0Fi1 5 MP</td><td>Nikon Corporation, JP</td><td></td><td></td></tr><tr><td>Osmium Tetroxide (OsO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Agar Scientific Ltd, GB</td><td>R1019</td><td>Danger: Fatal if swallowed, in contact with skin or if inhaled. Causes severe skin burns and eye damage Wear double protective gloves, protective clothing, eyes and face protection.</td></tr><tr><td>Pipette and pipette tips</td><td>Thermo Scientific, FI</td><td>KJ16047</td><td></td></tr><tr><td>Sodium dihydrogen phosphate dihydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt; 2H&lt;sub&gt;2&lt;/sub&gt;O) pure</td><td>PENTA, CZ</td><td>13472-35-0</td><td></td></tr><tr><td>Syringe 10 ml</td><td>Ecoject, DE</td><td>20010</td><td></td></tr><tr><td>Toluidine blue, general purpose grade</td><td>Fisher Scientific, GB</td><td>2045836</td><td></td></tr></tbody></table>

assessing structural traits in triticum aestivum and zea mays for c3 and c4 photosynthetic differentiation using free-hand and semi-thin sections

与 C3 机制相比，C4 光合作用的效率更高，源于其将 CO2 浓缩在束鞘细胞中的能力。C4 光合作用的有效性和内在水分利用效率与叶肉和束状叶细胞的份额、束状叶鞘的大小和密度以及束状鞘状细胞的大小、密度和细胞壁厚度直接相关。可以使用常规光学显微镜对徒手和半薄切片进行这些性状的快速显微镜分析，通过识别和检查特定细胞类型提供有关 C4 作物光合效率的宝贵信息。此外，还显示了影响解剖测量和细胞类型诊断的手绘和半薄片制备错误，以及如何避免这些错误。这种显微镜方法提供了一种有效的方法来收集对环境变化的光合适应的见解，并有助于快速筛选作物以适应未来的气候。

图 1A 显示了用于新鲜切片和光学显微镜检查的叶子切片的正确方向。使用单面剃须刀和牙科蜡片切割新鲜切片的方法如图 1B 所示。生成的部分如图 1C 所示。
图 2 显示了 C3 植物 T. aestivum 和 C4 植物 Z. mays 的代表性叶子的徒手切片。在叶脉周围可以看到束鞘?...

作者聚焦:了解气候适应型作物植物结构-功能关系的创新方法

评估 C3 和 C4 叶横截面之间的解剖学差异有助于了解光合作用效率。本文描述了徒手和半薄叶横截面的制备和检查，以及作物物种 Triticum aestivum 和 Zea mays 的准备注意事项。

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

The enhanced efficiency of C4 photosynthesis, compared to the C3 mechanism, arises from its ability to concentrate CO2 in bundle sheath cells. The ...

assessing-structural-traits-triticum-aestivum-zea-mays-for-c3-c4

Research

JoVE Journal

Biology

Assessing Structural Traits in Triticum aestivum and Zea mays for C3 and C4 Photosynthetic Differentiation Using Free-hand and Semi-thin Sections

894 次观看.  Estonian University of Life Sciences. 我们的研究重点是植物结构、功能关系。我们的目标是更好地了解不同植物功能类型的光合作用的局限性，以及如何改善它们以用于气候适应型作物。新技术和软件成本高昂，而且通常无法获得，这需要一定程度的试验和创新才能在对不同的植物功能类型进行成像时实现相同的分辨率。鉴定区分 C3 和 C4 叶的解剖结构，特别是在显微镜检查的准?...

视频: 作者聚焦:了解气候适应型作物植物结构-功能关系的创新方法

RGB and Spectral Root Imaging for Plant Phenotyping and Physiological Research: Experimental Setup and Imaging Protocols

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop cultivars against environmental stresses. An experimental protocol is presented for RGB and hyperspectral imaging of root systems. The approach uses rhizoboxes where plants grow in natural soil over a longer time to observe fully developed root systems. Experimental settings are exemplified for assessing rhizobox plants under water stress and studying the role of roots. An RGB imaging setup is described for cheap and quick quantification of root development over time. Hyperspectral imaging improves root segmentation from the soil background compared to RGB color based thresholding. The particular strength of hyperspectral imaging is the acquisition of chemometric information on the root-soil system for functional understanding. This is demonstrated with high resolution water content mapping. Spectral imaging however is more complex in image acquisition, processing and analysis compared to the RGB approach. A combination of both methods can optimize a comprehensive assessment of the root system. Application examples integrating root and aboveground traits are given for the context of plant phenotyping and plant physiological research. Further improvement of root imaging can be obtained by optimizing RGB image quality with better illumination using different light sources and by extension of image analysis methods to infer on root zone properties from spectral data.

An experimental protocol is presented for assessment of soil grown plant root systems with RGB and hyperspectral imaging. Combination of RGB image time series with chemometric information from hyperspectral scans optimizes insights into plant root dynamics.

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

Better understanding of plant root dynamics is essential to improve resource use efficiency of agricultural systems and increase the resistance of crop ...

科研

环境科学

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

生物学

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

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

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

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

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

与气孔导度，以叶功能性状

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

A Simple Dry Sectioning Method for Obtaining Whole-Seed-Sized Resin Section and Its Applications

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are significantly different among different regions of seed. In order to view the morphologies of cells, starch granules and protein bodies clearly, and quantitatively analyze their morphology parameters accurately, the whole-seed-sized section is needed. Though the whole-seed-sized paraffin section can investigate the accumulation of storage materials in seeds, it is very difficult to quantitatively analyze the morphology parameters of cells and storage materials due to the low resolution of the thick section. The thin resin section has high resolution, but the routine resin sectioning method is not suitable to prepare the whole-seed-sized section of mature seeds with a large volume and high starch content. In this study, we present a simple dry sectioning method for preparing the whole-seed-sized resin section. The technique can prepare the cross and longitudinal whole-seed-sized sections of developing, mature, germinated, and cooked seeds embedded in LR White resin, even for large seeds with high starch content. The whole-seed-sized section can be stained with fluorescent brightener 28, iodine, and Coomassie brilliant blue R250 to specifically exhibit the morphology of cells, starch granules, and protein bodies clearly, respectively. The image obtained can also be analyzed quantitatively to show the morphology parameters of cells, starch granules, and protein bodies in different regions of seed.

This technique allows for the fast and simple preparation of whole-seed-sized resin section for the observation and analysis of cells, starch granules, and protein bodies in different regions of the seed.

获得全种子尺寸树脂部分的简单干截块方法及其应用

The morphology, size and quantity of cells, starch granules&#160;and protein bodies in seed determine the weight and quality of seed. They are ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

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

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

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

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

通过表皮表型评分 鉴定 参与气孔发育的基因

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

Imaging and Analysis for Quantifying Maize (Zea mays) Abiotic Stress Phenotypes

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic stresses. However, traditional methods of measuring plants can be time-consuming, inaccurate, and destructive, particularly when assessing traits such as biomass and height on an individual basis. Image-based analysis offers an opportunity to reduce labor, improve accuracy and precision, and enable repeated measurements of the same plant over multiple time points. While high-throughput phenotyping facilities provide a solution, they are often prohibitively expensive and limited in availability worldwide. A method is described for capturing plant images using low-cost single-board computers and digital cameras within a photo studio setup, accompanied by an image analysis pipeline based on the free and open-source package PlantCV. This method offers a fast and accurate protocol for image capture, labeling, and transfer, along with best practices for ensuring high-quality image acquisition. Through image analysis, more than 10 phenotypes were quantified for all images in the experiment simultaneously, including traits such as plant size, height, and color. These methods were used to characterize the response of the Zea mays (maize) inbred variety B73 to heat, drought, and combined heat and drought abiotic stress conditions.

A method is presented for low-cost imaging of plants to measure phenotypes, along with best practices for image capture and an image analysis pipeline for quantifying plant traits. These methods were applied to measure maize (Zea mays) phenotypes under heat, drought, and combined abiotic stress conditions.

定量玉米 （Zea mays） 非生物胁迫表型的成像和分析

Measuring quantitative plant phenotypes, or traits, is essential for understanding plant responses to environmental factors, including biotic and abiotic ...

使用徒手和半薄切片评估小麦和 Zea mays 的 C₃ 和 C₄ 光合作用分化的结构特征

副本

摘要

探索更多视频

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

使用油脂介孔结构的测定结晶膜蛋白

组织化学染色

与气孔导度，以叶功能性状

混合式切割:用于顽拗植物组织样品的改进切法

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

获得全种子尺寸树脂部分的简单干截块方法及其应用

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

通过表皮表型评分鉴定参与气孔发育的基因

定量玉米（Zea mays）非生物胁迫表型的成像和分析

使用徒手和半薄切片评估 小麦和 Zea mays 的 C3 和 C4 光合作用分化的结构特征

副本

摘要

探索更多视频

RGB 和光谱根成像植物表型和生理研究: 实验装置和成像协议

使用油脂介孔结构的测定结晶膜蛋白

组织化学染色

与气孔导度，以叶功能性状

混合式切割:用于顽拗植物组织样品的改进切法

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

获得全种子尺寸树脂部分的简单干截块方法及其应用

用于解释霉菌异养植物组织中真菌定植和种子共生萌发的显微镜技术

通过表皮表型评分 鉴定 参与气孔发育的基因

定量玉米 （Zea mays） 非生物胁迫表型的成像和分析

使用徒手和半薄切片评估小麦和 Zea mays 的 C₃ 和 C₄ 光合作用分化的结构特征

通过表皮表型评分鉴定参与气孔发育的基因

定量玉米（Zea mays）非生物胁迫表型的成像和分析