傅承认, Rhenac 绿色科技股份公司的支持, 通过本研究的部分。从 DFG (SFB TR175) 获得了资金。

此协议包含一些示例, 说明如何分析A. 南芥植物的生长行为。
1. 准备
<ol><li>在开始之前, 仔细计划一下需要多少种植物来进行实验的可靠统计分析, 然后准备适当数量的花盆。 注意: 总是允许一些种子可能不会发芽。</li>
	<li>使用不同的单独可编程 LED 水平的气候室来比较在相同的一般环境条件 (材料表) 下的植物生长。</li>
</ol>2. 植物生长和 LED 灯的设置
<ol><li>准备适当的数量 (取决于不同的条件和/或突变体要分析) 的 6 x 7 cm 盆与土壤和植物在每一个单一的种子。 注: 在这种情况下, 每种条件下播种了36株植物。</li>
	<li>Vernalize 在4° c 的两天。</li>
	<li>设置相对湿度为 65%, 温度为22° c/16 ° c, 为16小时/8 小时夜间循环。将所有级别的光照调整为200µM/cm2/秒1的强度。通过在高潮室前面的触摸屏上输入相应的值进入一个程序来做到这一点。要比较四种不同的光源质量, 将指示灯设置为以下参数, 如表1所示。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>Identifyier</td>
			<td>395毫微米 [%]</td>
			<td>440毫微米 [%]</td>
			<td>3 K [%]</td>
			<td>660毫微米 [%]</td>
			<td>770毫微米 [%]</td>
		</tr>
<tr>
<td>阳光"</td>
			<td>100</td>
			<td>11</td>
			<td>100</td>
			<td>15</td>
			<td>100</td>
		</tr>
<tr>
<td>红色和蓝色 (RB)</td>
			<td>100</td>
			<td>15</td>
			<td>25</td>
			<td>10</td>
			<td>100</td>
		</tr>
<tr>
<td>蓝色 (B)</td>
			<td>100</td>
			<td>15</td>
			<td>25</td>
			<td>2</td>
			<td>25</td>
		</tr>
<tr>
<td>红色 (R)</td>
			<td>90</td>
			<td>2</td>
			<td>25</td>
			<td>10</td>
			<td>100</td>
		</tr>
</tbody></table>表 1: 发光二极管发出的光强度的组成
<ol start="4"><li>连续监测频谱输出,例如使用内置校准光谱仪。</li>
	<li>把植物放在气候室的不同层次上, 并保持它们覆盖在一个透明的顶端, 直到发育良好的子叶可见。确保植物充分浇水。</li>
	<li>根据植物在所选择的条件下的生长速度, 每两天对植物进行眼部和摄影的监测和记录. 确保使用三脚架为相机, 以确保相机和对象之间的相同距离的所有图片, 从而使比较。适当时使用缩放栏。 注: "DAS" 一词 (播种后的天数) 指的是种子的实际种植, 包括春。</li>
</ol>3. PSII 产量的测定
<ol><li>使用脉冲调制 fluorimeter。设置在适当的距离从植物的相机头, 使完整的玫瑰花可以看到在现场的窗口。</li>
	<li>启动软件时, 将出现 "选择设备" 窗口。勾选 "迷你", 然后 "ok"。在下一个弹出窗口中选择颜色 "蓝色"。单击 "确定"。 注: 脉冲调制荧光测量灯自动开启。在显示器上, 图像窗口显示荧光参数 Ft。放置在照相机下的植物现在可以被看作是橙色的图像。</li>
	<li>要集中图像和/或选择特定区域的植物, 切换到现场视频的滴答作响的 "现场视频" 框, 并打开调整环的物镜。单击右上角的 "退出" 框, 退出 "实时视频" 窗口。</li>
	<li>为了测量光合参数, 定义一个感兴趣的区域 (君)。使用默认设置, 它是一个自动出现在屏幕中间的圆圈。它旁边的红色方框代表了所有像素的平均 Ft 值。通过从右侧面板的 "从" 框中选择相应的形式来定义适当的君。单击 "添加" 选项卡, 并在叶区域内放置圆圈。每叶重复五次。</li>
	<li>在制造商提供的默认值 (表 2) 中维护设置 (右侧的选项卡)。</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>光</td>
			<td>国际.</td>
			<td>频率</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>1</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>法.光</td>
			<td>国际.</td>
			<td>宽度</td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>8</td>
			<td>0</td>
			<td></td>
		</tr>
<tr>
<td>图像校正</td>
			<td>迷你</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>图像转换</td>
			<td>电池</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td>16.7V</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>获得</td>
			<td>5</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>阻尼</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>Sat 脉冲</td>
			<td>国际.</td>
			<td>不</td>
			<td>间隔 s</td>
		</tr>
<tr>
<td></td>
			<td>8</td>
			<td>1</td>
			<td>30</td>
		</tr>
<tr>
<td>慢感应</td>
			<td>延迟 s</td>
			<td>时钟 s</td>
			<td>持续时间 s</td>
		</tr>
<tr>
<td></td>
			<td>40</td>
			<td>20</td>
			<td>315</td>
		</tr>
<tr>
<td>Absortivity</td>
			<td>红色增益</td>
			<td>红色强度</td>
			<td>近红外强度</td>
		</tr>
<tr>
<td></td>
			<td>340</td>
			<td>25</td>
			<td>13</td>
		</tr>
<tr>
<td>显示</td>
			<td>颜色</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>PS 限制</td>
			<td>50</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>肼.Ref. 君</td>
			<td>1</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>调频因数 (刻度)</td>
			<td>1030</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 2: 由制造商提供的 PAM 测量的默认设置。
<ol start="6"><li>应用饱和光闪光进行光合参数的荧光猝灭分析 (饱和状态脉冲) 测量。在此之前, 通过测量暗适应植物的最小和最大荧光率来确定淬火系数。为此, 将植物放在黑暗中 (如在抽屉或黑盒子里的), 使其处于最小值。然后把植物放在相机头下, 检查盒子的测量和 ML (测量光), 在下面的行中选择 "Fv/fm", 通过计时进入圆圈, 并点击 Fo, 调频在屏幕的底部。 注意: Fo/Fm 代表了一个暗适应的植物的 PSII 产量。因此, 应用光后测量的值是归一化的。当前的 Fo/Fm 测量将保持直到新的记录被激活。由饱和脉冲决定的所有 F 和 fm 的值与 Fo/调频和淬火参数进行了相应的计算。</li>
	<li>在 "报表" 选项卡中查找这些结果, 并检查与实验相关的所有右侧框 (e. g。Y (II),/qn,等)。</li>
	<li>将结果导出到分析软件e. g。Excel 通过单击左上角的 "导出" 按钮并保存在相应的文件名下。生成至少五 AOIs 每叶和测量同一个植物的几个叶子 (不要忘记对短期黑暗适应植物在每次测量以后), 并且几棵植物从一个情况, 可以然后统计地被评估。</li>
	<li>为统计分析产生平均值和标准偏差从所有 AOIs 和至少三独立植物为所有时间点或情况并且执行学生的 t 测试评估数据是否是重要的12。当 p 值低于0.05 时, 将对数据进行显式不同的计算。</li>
</ol>4. 气孔密度的测定
<ol><li>在玻璃培养皿中收集三个完全膨胀的玫瑰花叶, 每个条件为70% 乙醇, 提取叶绿素。在室温下或在4° c 贮存时, 在该溶剂中孵育。</li>
	<li>为充分清除从颜料, 孵育在水合氯醛溶液中 (水合氯醛: 水: 甘油 = 8: 2: 1 瓦特/伏/w) 直到叶子出现完全白色。</li>
	<li>在40X 放大倍数下, 取背面表面的差分干涉显微镜 (DIC) 图像。计数气孔在视野和外推的帮助下规模酒吧的气孔每毫米。对每个条件至少4叶重复此步骤。通过计算一个条件下的所有叶的平均值来进行统计分析, 并由此计算平均误差。</li>
</ol>5. 鲜重的测定
<ol><li>去除所有叶子包括叶柄从六棵植物的花环与刀片的每个条件。立即称量所有叶子, 并按照上述数据进行统计分析。</li>
</ol>6. 玫瑰叶面积的测定
<ol><li>使用图片从八植物每个条件分析叶面积图形。将图片合并为单个图像中的一个条件, 然后另存为 *. jpeg 或 *. tiff。</li>
	<li>下载相应的软件 (材料表)。 注意: 当然, 可以应用任何其他能够执行此任务的程序。</li>
	<li>打开一个图像文件。选择工具 "自由手选择"。包围叶包括一个莲座丛的叶柄。单击 "分析-设置测量" 并检查 "区域"、"最小 & 最大灰度值"、"集成密度" 和 "平均灰度值"。选择适当的小数位, 最好是两个或三, 然后单击 "确定"。</li>
	<li>若要获取 mm2而不是像素, 请使用 "设置比例" 命令。应用直线选择工具使行选择与已知距离相对应,例如从图像的刻度栏轻松计算的离开直径, 然后打开 "设置比例" 对话框, 然后输入此定义的距离和单位测量.然后返回 "分析", 然后单击 "测量"。</li>
	<li>出现一个新窗口, 标题为 "结果", 其中包含当前花环的相关数据。重复此过程与所有植物在图像和初始化面积测量为每一个新的工厂与 ctrl+ m。 注: 对于具有非重叠叶子的较小的植物, 可以使用 "魔杖工具" 使过程变得更容易、更快。一旦树叶开始互相覆盖, 这个工具就不会给出可靠的结果。</li>
	<li>或者, 切断所有的叶子, 并把它们放置在一个概述图片可以采取的方式, 然后使用魔杖工具。考虑到使用这种方法时, 需要更多的植物。</li>
	<li>在 "结果" 窗口中选择 "文件"-"另存为", 并在计算机目录中生成适当的文件名和位置。该文件将以 Excel 格式自动保存。</li>
</ol>7. RNA 的准备
<ol><li>每种情况下从十株植物中收获三样品。根据制造商的说明, 使用植物 rna 提取试剂盒提取总 rna。使用 bioanalyzer 测定 RNA 的浓度、纯度和完整性。然后, RNA 可以用于下游应用, 如 qRT PCR 或基因表达分析, 由如RNASeq13。</li>
</ol>

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作者没有什么可透露的。

研究植物生长的第一步是根据所需条件建立气候室。通过将所有变量键入相应软件的程序掩码 (图 1A), 可以轻松完成此操作。在这一步, 许多修改可以通过改变光的制度和/或温度来实现。确保不断监视温度、湿度和光照条件 (图 2), 以防止技术故障破坏实验。这是获取可重现结果的关键点。虽然这种设置提供了许多变量, 可以灵活地调整, 但它有其局限性。目前可用的 LED 灯无法模拟阳光 100%, 气候室内的气候条件永远无法完全反映出15外部发生的情况。
与广泛使用的荧光灯相比, LED 灯更多功能, 需要更少的能量, 而且几乎不显示热辐射。这些优势导致了大工业的室内农业装备气候室和温室与 led16。考虑到这一领域的巨大成功, LED 技术肯定会找到更多的应用。
当观察表型, 特别是对叶面积的测定时, 重要的是要考虑到在较老的植物叶子重叠 (图 3)。因此, 整个花环的图形化评价往往不精确。在这种情况下, 它是更准确的切掉所有的树叶, 并从那里去。
在不同条件下, 对生长行为的评价, 特别是生长发育的差异, 取决于样本大小是否足够。在这项研究中, 至少有六株植物用于测定例如光合产量 (图 5)、鲜重 (图7) 和叶面积 (图 8A), 但在该研究开始时种植了30个人种子, 以确保首先, 足够的种子发芽, 第二, 可以选择 "典型的" 植物。即使在相同的种群中, 在相同的条件下, 在同一托盘中的单盆中, 也有不同的表型. 这当然反映在统计分析期间的标准偏差中, 但是当观察到小的统计错误 (图 8B) 时, 数据的解释通常更可靠。
通过 PAM (图 4,图 5) 可以对几个参数进行光合性能的测量。在这种情况下, 重点是 PSII 屈服 Y (II) 为例, 但也可以确定例如非光化学猝灭、调节和非调节的能量耗散或光缓解的量子屈服。重要的是选择至少五 AOIs 每叶均匀分布在叶片表面, 然后测量至少六叶从不同的植物。这种方法的缺点是不能检测到对 PSI 的任何影响;为此目的, 需要不同的设备。

在二十多年的时间里,拟南芥一直是分子时代植物研究人员的模型有机体。几个特点使这个小代表的芸薹家族的理想候选基因和分子研究: 它有一个相对较小的基因组, 只有五染色体 (相比,如烟草烟草与24染色体) 和它的基因组在 2000年1中被完全测序。A. 南芥可以很容易地通过农杆菌病感染2进行基因改造, 甚至能够使用最新的基因工具, 如 CRISPR/Cas3。虽然小, 生长周期是足够快, 使生物化学实验是可行的, 在需要更大量的材料。植物生长在琼脂板材或在土壤和可以甚而耕种作为液体文化4。拟南芥可在高潮控制的碗柜中生长,例如, 来自波斯富, 在气候室或温室中。为了能够比较生长行为和分析突变体的表型, 这是至关重要的提供可重现性和同时灵活的增长条件5。根据将被解决的科学问题你可能需要不同的温度和恒定的光条件, 不同的光强度或不同的光质量在同一温度。光是植物生长的一个非常关键的参数, 它的影响在不同的方法中经常被研究6。为了确保获得的数据的可重复性和可比较, 确保稳定的输出并应用相同类型的光源是至关重要的。
温室和气候室通常的光源由钠蒸气或荧光灯组成, 它们促进了植物的生长, 但有几个缺点。首先, 他们的年龄随着时间的推移, 改变了光谱输出不仅在强度, 但在质量 (自己的观察)。然而, 只有强度通常是连续监测的, 所以光质量的变化可能会被忽视, 但仍然有显著的影响。第二, 两种类型的灯具在较高的光照强度下产生热量, 这本身对植物生长有深刻的生理影响, 并可能掩盖任何光的依赖性效应。第三, 这些光源的光谱输出是不变的, 很不像自然的阳光7。所有这些缺点都已在 led 的情况下被克服8,9,10,11。它们的寿命很长, 几乎没有任何变化, 即使在极高的光强下也不会产生废热, 它们在光谱输出方面非常灵活。
在这里, 我们说明了如何建立一个气候室, 分别为红色, 蓝色和白色光的 LED 灯, 并遵循不同的植物生长参数随着时间的推移。我们测量新鲜的重量, 叶面积, 气孔密度, 和光合性能。同时, 我们也展示了正确设置统计评价的重要性。

<table>
	<tbody>
		<tr>
			<td>Climatic chamber equipped with LED panels</td>
			<td>Rhenac Green Tec AG</td>
			<td></td>
			<td>These chambers are custom made.</td>
		</tr>
		<tr>
			<td>Spectrometer&#160;</td>
			<td>OceanOptics</td>
			<td>USB-650</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging PAM</td>
			<td>Walz</td>
			<td>IMAGING-PAM M-Series</td>
			<td>There are several suitable models depending on the broader use.</td>
		</tr>
		<tr>
			<td>Microscope+ 40x objective</td>
			<td>Leica&#160;</td>
			<td>DM1000</td>
			<td>Other companies also produce suitable microscopes.</td>
		</tr>
		<tr>
			<td>Software ImageJ</td>
			<td></td>
			<td></td>
			<td>Free download from website</td>
		</tr>
		<tr>
			<td>Plant RNA extraction kit</td>
			<td>Qiagen</td>
			<td>74903</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyser</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>Needs an additional computer</td>
		</tr>
	</tbody>
</table>

analysis of arabidopsis thaliana growth behavior in different light qualities

植物生物学家经常需要观察他们所选择的物种的生长习性。为此, 植物需要恒定的环境和稳定的光条件, 这是最好的数量和质量变量, 以便在不同的设置可以进行研究。这些要求由具有发光二极管 (LED) 灯的气候室满足, 它可以与荧光灯形成不同的波长。发光二极管是能量守恒, 即使在光强度下也几乎不发出热量, 这通常构成了其他光源的问题。该协议提供了一步一步的指导, 如何规划一个气候室装有可变 LED 灯, 以及描述几种方法, 深入分析的增长表型。根据试验装置的不同特性, 可以观察和分析生长中的植物。在这里, 我们描述了如何确定新鲜的重量, 叶面积, 光合活性和气孔密度。我们证明, 为了获得可靠的数据和得出有效的结论, 必须使用足够数量的个人进行统计评估。用太少的植物进行这种分析会导致高的统计误差, 因此对数据的解释不太清楚。

观察和分析植物生长, 特别是表型从突变植物依赖于稳定和可再生的环境条件。这些可以在气候室提供。光量, 特别是质量的关键取决于所使用的光源, 这项研究是由 LED 灯提供的。
图 1显示了一个装有 LED 面板的气候室的示例。图 1A显示了控制面板的屏幕截图, 可以调整所有的气候和光照条件。在24ĥ二十不同的时间框架可以设置。在这个例子中, 有 16 light/8 的长日条件已经被编程。该会议厅具有四个级别, 可以分别编程, 以便在完全相同的环境条件下, 可以研究四不同光照背景下的植物生长。左上角的水平被设置为一个光谱输出模仿日光尽可能多的技术上, 右上水平代表高架红 (660 nm) 和蓝光 (440 nm) 与减少白光 (3K)。左下一级设置为升高的蓝光和右下水平, 以红色为主光。图 1B将不同设置中的 led 显示为概述 (中间面板) 和相应的缩放 (外部小面板)。光的质量差异可以很容易地看到眼睛。
内置光谱仪不断测量、监测和调整频谱输出。图 2显示了左上角1.1 的频谱, 它被设置为模拟太阳光。与标准萤光灯泡相比, UV 和蓝光的部分更高7。
在图 3中, 从所有四条件10、13和17天的A. 南芥中, 分别描述了播种后的例子。所有的植物都是从同一距离拍摄的, 安装在三脚架上的摄像头。刻度线代表1厘米。10天后, 在大小或颜色上没有太大的区别可以看出, 但在17天后, 红灯下的快速增长是显而易见的。除了这种视觉分析, 还进行了一些生理分析。
图 4遵循 PAM 测量的不同步骤, 它分析了如光合能力。在图 4A中, 显示了实时视频的屏幕截图, 这是将该设备集中在一起以确保测量的最佳质量的设置。而不是集中在整个植物, 你也可以选择一个单一的叶子来分析。图 4B演示了在实际测量开始之前, 一个暗适应的工厂目前的荧光产量 Ft。在这种情况下, 选择了五圆的感兴趣区域 (AOIs)。每个君旁边的红色方框中的数字直接给出了数值结果, 也可以用表的形式保存。为了开始测量光合参数, Fm 需要设置。在图 4C中描述了这样做后的 Ft 屏幕快照。请注意, 现在按钮 "Fo, Fm" 已经不再活跃。要开始新的度量, 需要单击 "新记录" 以清除以前的规范化。最后,图 4D显示了在发出饱和光脉冲 ("SAT 脉冲") 后有效的 PSII 量子屈服 Y (II)。示例数据的量化显示在图 5中。在阳光下生长的植物200µM/厘米2/秒1 (图 5A) 分别进行了12、21和28天的播种。我们的数据表明, 在三周的植物叶片中, PSII 的产量明显高于12天。28和12天之间的差异仍然很大, 但 p 值较高。在图 4B中, 比较了从不同光照质量中生长两周的植株的 PSII 产量。有趣的是, 在光中含有高部分蓝光的永久性生长会导致 PSII 的产量显著提高。在丰富的红光下种植的植物也有类似的效果, 但增幅略低。
不同的光质量显示出影响气孔发育14。因此, 研究了气孔密度。图 6演示了颜料提取后的叶子的外观。单表皮细胞可以很好的区分, 气孔可以很容易计数。在图中, 单个气孔由星号表示。有关不同光照环境下植物气孔密度的详细数据, 可在其他地方找到9。
除了目视检查 (图 3) 外, 新的重量还提供了一个很好的衡量生长进度的指标。在本例中, 在8、10和12天后的 "阳光" 下生长的植物分别被称重。这些数据的统计评估可以在图 7中看到。正如所料, 新的重量随着时间增加。
除了新鲜的重量, 叶面积是一个很好的衡量增长。在这里, 植物的开发是从 10, 13, 和17天后播种 (图 8A)。为了获得可靠的统计数据, 对至少六个别植物进行了例行评估。为了证明高样本大小的重要性, 计算了两个和六种植物分别分析平均值的百分比误差 (图 8B)。这意味着标准偏差的百分比关于平均值被确定了。很明显, 在小样本大小的情况下, 误差是 5-10% 以上的情况下更高的样本大小。通过增加被评估的植物的数量, 错误可以被最小化, 使解释数据更加清楚。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57152/57152fig1.jpg"> 图 1: 指示灯提供不同的光源质量。A)从 LED 箱的控制面板截图。日长设置为 16 h (右上角), 光照强度设置为200µmol cm-2 s-1。光质量在所有四水平上是不同的: 1.1 代表一个光谱作为相似于阳光技术上可能, 1.2 代表红色和蓝色波长 (铷) 光的高百分比, 2.1 主要地被设置蓝色 (B), 2.2 代表主要红色光 (R)。B)中间的面板显示了所有级别的概览;外部面板在更高的缩放中显示各个级别。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57152/57152fig2.jpg"> 图 2: 模拟阳光背景下的波长频谱.一个截图从内置光谱仪在 LED 室显示, 这是定位在1.1 级。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57152/57152fig3.jpg"> 图 3: 一周内工厂开发.代表A. 南芥从10、13和 17 DAS 的所有四光照条件下进行植物。植物用三脚架上的数码反光相机拍照。缩放条代表所有图像 1 cm。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57152/57152fig4.jpg"> 图 4: 来自A. 南芥植物的 PAM 测量的代表性步骤的截图.A)从 "实时视频" 视图中可以调整图像焦点的屏幕截图。B)在任何光脉冲的应用之前, 电流荧光的产生。C)在设置了饱和光脉冲后产生的 PSII 量子屈服的电流荧光率. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57152/57152fig5.jpg"> 图 5: 影响 PSII 量子屈服 (YII) 的图形表示.A)数据从植物 12, 21, 和28天播种和生长在200µmol/cm2/s1下的模拟太阳光 ("阳光") 受到 PAM 分析的统计评价。显示的平均值为五植物和五 AOIs 每天。一个星号表明与 p 值 < 0.05 相比, 12 天, 两个星号表示非常显著的差异与 p 值 < 0.02 根据学生的 t 测试。b)数据分别从200µmol/cm2 /s1的模拟阳光 (SL)、富集蓝 (B) 或红色 (R) 光中进行统计评估。显示的平均值为五植物和五 AOIs 每天。与 "阳光" 相比, 计算出了显著的差异。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57152/57152fig6.jpg"> 图 6: 在A. 南芥叶的背面的气孔的代表图像.如上所述的叶子, 在40X 放大倍率的光显微镜下进行目测分析。气孔计数在至少4叶的可见面积每个条件。这张照片是用与显微镜博思连接的数码相机拍摄的。每个毫米的气孔数是借助于刻度棒来计算的。星星表示一个气孔。刻度栏代表200µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57152/57152fig7.jpg"> 图 7: 从A. 南芥植物生长在模拟 sunlight/200 µmol/cm2/s1中的新重量的图形表示。玫瑰花叶从植物八, 十, 和十二天后播种。描述了每天六株植物中 mg 的平均值。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57152/57152fig8.jpg"> 图 8: 在不同光照条件下生长的植物的A. 南芥叶面积的统计评价.a)从以10、13和17天为基地的. 南芥的叶面积以 ImageJ 和数据从n = 6 植物被统计地被评估了图解地确定了。从每个条件的所有六莲座丛的叶面积总结和除以六, 以获得平均值。使用此值, 计算标准偏差, 并由误差线表示。B)叶区域以图形方式确定, ImageJ 和数据分别来自n = 2 或n = 6 株植物, 统计分析为小组 A 描述。然后以图形方式计算和描述平均值的百分比误差。绿色条形图显示来自分析n = 6 的百分比错误, 来自n = 2 植物的蓝色条形图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57152/57152fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities at JoVE.com

不同光质下拟南芥生长行为分析- 中国优秀硕士学位论文全文数据库生物学 |视频

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

在这里, 我们提出了一个研究植物生长行为的协议, 特别是表型以可重现的方式。我们展示如何提供可变和在同一时间稳定的光条件。正确的分析取决于足够的样本数和有效的统计评价。

不同光照条件下的拟南芥生长行为分析

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...

analysis-arabidopsis-thaliana-growth-behavior-different-light

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Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

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18.8K 次观看.  Ludwig-Maximilians-Universität. 在这里, 我们提出了一个研究植物生长行为的协议, 特别是表型以可重现的方式。我们展示如何提供可变和在同一时间稳定的光条件。正确的分析取决于足够的样本数和有效的统计评价。

视频: Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

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生物学

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

从14日龄的幼苗组织原生质体的分离拟南芥</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

调查组织和器官特定的光敏色素反应，使用流式细胞仪辅助细胞类型特定的表达谱拟南芥</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

花香浸改造拟南芥检查 pTSO2：β-葡萄糖醛酸酶报告基因的表达

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

拟南芥极地甘油脂分析薄层色谱（TLC）再加上气相色谱（GLC）

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

种皮床品检测到基因探索<em&gt;体外</em&gt;如何控制胚乳种子萌发的<em&gt;拟南芥</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

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 ...