Name: high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry
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Description: Watch this Scientific Journal Video about High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry at JoVE.com

这项工作得到了研究项目“实现提高光合作用效率”（RIPE）的支持，该项目由比尔和梅琳达·盖茨基金会，粮食及农业研究基金会以及英国外交，英联邦和发展办公室资助，拨款号为OPP1172157。

1. 种子种植
<ol><li>选择肥沃，排水良好但不是沙质土壤的田地，pH值接近6.5。用锄头对地面进行评分，标记出间距为0.75 m的 1.2 m 行图。</li>
	<li>在土壤温度在25至30°C之间的生长季节开始时，沿每个地块在3厘米深处种植50粒 G.max cv. IA3023。 注意：为了筛选遗传多样性，预计会生长和比较多种不同的基因型。每个基因型植物2-5行，以随机块设计排列。应考虑气候条件是否适合大...

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仔细选择和处理叶盘对于获得可靠的NPQ测量值至关重要。首先，对组织的损伤，例如用镊子粗暴处理，将引入应力，导致光合作用的最大量子效率值较低。非胁迫植物的 Fv/Fm 值通常约为0.8318，显著下降表明光合性能下降9。然而，在田间条件下生长的植物通常经历轻微的胁迫，因此根据先前的研究，将 FvFm...

光合作用包括光吸收、初级电子转移、能量稳定以及光合产物的合成和传输1.了解每个步骤对于指导提高作物光合作用效率的努力至关重要。光影响光合作用的速率，需要以光子的形式平衡能量供应，并需要减少等效物。当供应超过需求时，例如在高光下或气孔闭合引起的CO2 固定减少期间，还原功率的积聚增加了活性氧形成的可能性，有可能损坏光合装置并损害电子传输。因此，为了防止损伤，植物已经发展出几种光保护机制，包括活性氧的解毒和激发叶绿素态（NPQ）的非光化学淬灭2。
在野外环境中保持高光合作用率具有挑战性。季节性和昼夜变化，以及风引起的叶片运动和瞬时云层覆盖等环境波动，导致植物光合作用3所接收的光量和强度发生变化。NPQ可以消散多余的光能，并有助于防止光损伤，同时允许在高光4下持续光合作用。然而，在高光到低光跃迁期间，长时间的NPQ继续耗散可用于碳减排的能量5。因此，加速NPQ的松弛可以提高光合作用6的效率，使NPQ松弛成为作物改良的有吸引力的靶点。
脉冲幅度调制叶绿素荧光（PAM）分析可用于根据可测量参数计算NPQ（补充表1 和 补充表2）7，8，9。本文重点介绍如何确定田间植物中的NPQ弛豫率，以筛选种质中的自然变异。然而，PAM叶绿素荧光测定法分析也可用于各种目的，适用于从藻类到高等植物的物种，并在其他地方进行了综述7，8，9。
在暗适应的叶子或细胞中，光系统II（PSII）反应中心是开放的，可以接收电子并且没有NPQ。打开低强度测量光可引发叶绿素荧光，同时避免电子通过PSII传输。在这种暗适应状态下记录的最小荧光由参数Fo描述。将高强度光脉冲施加到深色适应的叶子上可以迅速减少与醌A位点结合的醌的第一个稳定电子受体池。这暂时阻断了PSII反应中心的电子转移能力，然后说这些反应中心是封闭的，无法从水分裂中接收电子。通过使用较短的脉冲持续时间，没有足够的时间来刺激NPQ。所得叶绿素荧光相当于在没有NPQ的情况下可获得的最大值，或最大荧光F m。最小荧光和最大荧光之间的差异称为可变荧光，Fv。光系统II的最大光化学量子产率（Fv/Fm）是使用以下等式从这两个参数计算得出的：
Fv/Fm = （Fm-F o）/Fm
这可以提供光系统功能和应力的重要指标。打开光化（光合）光会刺激非光化学猝灭，随后应用饱和闪光可以测量适合光的最大荧光 Fm'。通过比较暗荧光和适应光的最大荧光之间的差异，可以根据斯特恩-沃尔默方程10计算NPQ：
NPQ = F米/联邦m' - 1
在高等植物中，氮磷脂被描述为由至少五种不同的成分组成，包括质量相关成分、qT、qZ、qI和qH。NPQ所涉及的确切机制尚不完全清楚。然而，在大多数工厂中，qE被认为是NPQ的主要成分。已经发现完全参与qE的关键因素包括质子梯度在类囊体膜上的积聚，光系统II亚基S11，12的活性，以及脱环氧化的叶黄素，动脉炎，叶黄素，特别是玉米黄质13。qE是所有NPQ组分中松弛最快的（<2分钟）14，因此qE的可逆激活对于适应不断变化的光强度尤为重要。NPQ弛豫的第二个较慢阶段（约2-30分钟）包括与状态转换相关的qT和qZ，涉及玉米黄质与中提黄质15的相互转化。NPQ的缓慢放松（>30分钟）可能包括光抑制淬灭（qI）16 和独立于光损伤17，18的过程，例如qH，其是由质体脂质体运筹蛋白19，20介导的PSII外周触角中的持续淬火。
在高光下，NPQ 会增加。随后转移到低光下可导致NPQ的下调。快速、中间和慢弛鬆相位的衰减可以在双指数函数15，21，22，23 的参数中捕获
NPQ = Aq1（-吨/τ1） + Aq2（-吨/τ2） + Aq3
双指数函数的理论基础是基于假设淬火器的一阶利用假设，包括qE（Aq1），qZ和qT的组合松弛（Aq2），相应的时间常数τq1和τq2，以及长期NPQ，其中包括qI和光损伤无关过程（Aq3）。因此，与缺乏理论基础的更简单的希尔方程相比，双指数函数提供了猝灭叶绿素荧光所涉及的多个连接生物过程的更真实的表示24。
NPQ可以使用各种市售的PAM荧光计25、26进行测量，从简单的手持设备27 到更先进的封闭系统28。然而，其中几种方法的局限性是通量相对较低，这使得在没有多个设备和研究团队的情况下筛选大量植物具有挑战性。为了解决这个问题，McAusland等人开发了一种基于切除的叶组织的程序，并用它来鉴定两个小麦品种29之间叶绿素荧光的差异。这种方法的吸引力在于，用一个设备从多个植物中提取的成像叶盘可以促进在一天内筛选数百个基因型。这使得评估NPQ弛豫的变化成为可能，作为全基因组关联研究的一部分，或者用于筛选有可能提高作物光合效率和最终产量的育种种群。
基于McAusland等人29的发现，我们使用叶盘的PAM叶绿素荧光分析来高通量筛选 甘氨酸max （G.max;大豆）中的NPQ弛豫率。该协议使用CF成像仪25，可与其他市售的封闭式PAM系统相媲美，例如流行的FluorCam26。通过用于调整样品的暗室，用户可以对96孔板，培养皿和小型植物进行成像。与单个植物的顺序分析相比，这种方法的主要优点是使用叶盘可提高吞吐量。在这里，我们提出了具有代表性的结果，以及一种在田间生长的植物中采样，测量和分析NPQ的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>24 well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>FB012929</td>
			<td>Country of Origin: United States of America</td>
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			<td>96 well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>FB012931</td>
			<td>Country of Origin: United States of America</td>
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			<td>Aluminum foil</td>
			<td>Antylia Scientific</td>
			<td>&#160;61018-56</td>
			<td>Country of Origin: United States of America</td>
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			<td>Black marker pen</td>
			<td>Sharpie</td>
			<td>SAN30001</td>
			<td>Country of Origin: United States of America</td>
		</tr>
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			<td>CF imager</td>
			<td>Technologica Ltd.</td>
			<td>N/A</td>
			<td>chlorophyll fluorescence imager 
			Country of Origin: United Kingdom</td>
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			<td>Cork-borer, 7mm</td>
			<td>Humboldt Mfg Co</td>
			<td>H9665</td>
			<td>Country of Origin: United States of America</td>
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			<td>FluorImager V2.305 Software</td>
			<td>Technologica Ltd.</td>
			<td>N/A</td>
			<td>imaging software 
			Country of Origin: United Kingdom</td>
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			<td>iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters</td>
			<td>Amazon</td>
			<td>&#160;B07P6XCTGV</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Marker stakes</td>
			<td>John Henry Company</td>
			<td>KN0151</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Paper scissors</td>
			<td>VWR</td>
			<td>82027-596</td>
			<td>Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis Company Inc.</td>
			<td>&#160;S3-594-6</td>
			<td>Semi -transparent flexible film 
			Country of Origin: United States of America</td>
		</tr>
		<tr>
			<td>Solid rubber stoppers</td>
			<td>Fisher Scientific</td>
			<td>14-130M</td>
			<td>Country of Origin: United States of America</td>
		</tr>
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high-throughput analysis of non-photochemical quenching in crops using pulse amplitude modulated chlorophyll fluorometry

光合作用在现代作物品种中没有得到优化，因此提供了改进的机会。加速非光化学淬火（NPQ）的松弛已被证明是提高光合性能的有效策略。然而，由于过度采样和从田间生长的作物植物收集的局限性，缺乏为改善NPQ而育种的潜力以及对NPQ松弛的遗传基础的完整理解。在之前的报告的基础上，我们提出了一种高通量测定法，用于使用脉冲幅度调制（PAM）叶绿素荧光测定法分析 甘氨酸max （大豆）中的NPQ弛豫率。在运输到实验室之前，从田间种植的大豆中取样叶盘，在实验室中用封闭的PAM荧光计测量NPQ弛豫。NPQ弛豫参数的计算方法是在从高光到低光转换后将双指数函数拟合到测量的NPQ值。使用这种方法，可以在一天内测试数百种基因型。该程序有可能筛选突变体和多样性小组在NPQ弛豫中的变化，因此可以应用于基础和应用研究问题。

图1A 描述了大田种植的大豆中NPQ的典型测量值。2021年夏季在伊利诺伊州厄巴纳（纬度40.084604°，经度-88.227952°）种植植物，并于6月5日播种。2021. 在播种30天后对叶盘进行采样，并使用提供的方案进行测量（表1）。计算每个叶盘的 Fv/Fm 和NPQ值（补充表4），并通过拟合双指数函数计算NPQ松弛参数（表2）。在初始饱和闪光以?...

Watch this Scientific Journal Video about High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry at JoVE.com

High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

该协议引入了一种高通量方法，用于通过脉冲幅度调制的叶绿素荧光测定法测量非光化学淬灭的松弛。该方法适用于田间生长的 甘氨酸max ，并且可以适应其他物种以筛选遗传多样性或繁殖种群。

使用脉冲幅度调制叶绿素荧光法对作物中的非光化学猝灭进行高通量分析

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of ...

该方法可以深入了解遗传多样性面板或育种群体中可用的非光化学淬火的遗传变异。该技术的主要优点是有可能在一天内筛选出大量基因型在非光化学淬灭中的变异。该协议针对甘氨酸max或大豆提出，但可以调整为分析可以从中收集升降盘的其他植物空间。对于第一次执行该协议的人来说，关键是处理提升盘以防止损坏或用水过度饱和海绵。首先，在发芽后30天在野外对植物进行取样。在24孔板的所有孔中填充1/3水平的蒸馏水。在盖子和板的侧面标记要取样的重复。将最年轻的全膨胀叶子放在植物的顶部，放在橡胶塞上。将两号洪堡软木螟穿过叶子并扭转以切割圆盘，避开中间肋骨。从同一片叶子中连续收集五个圆盘进行技术复制。使用棉签将叶盘从软木螟中挤出到24孔板的单孔中，并对相同基因型的不同植物重复此操作。检查所有叶盘是否漂浮在水中。如果没有，用棉签轻轻地将粘在井边的叶盘移动到漂浮的位置。继续从下图中取样光盘。盖上盖子，用半透明的柔性薄膜密封。在整个24孔板中完成采样后。将盘子存放在袋子、盒子或空冷却器中，避免阳光直射。在干净的实验室空间中，轻敲密封板的盖子，以在运输过程中将粘在盖子上的叶盘移开。打开薄膜并取下盖子。将叶盘从24孔板的第一个位置转移到新鲜的96孔板中，叶盘朝向孔底部的平直下方。将鼻吸器过滤器切成两半。将得到的过滤器浸入水中一半，然后轻拍纸巾以除去多余的液体。用叶盘将过滤器插入孔中以保持湿度。从24孔板的第一个位置取第二个叶盘，并将其面朝下放置在96孔板的下一个可用位置。将鼻腔过滤器的剩余一半浸入水中，将其轻拍在纸巾上，然后将其与第二个叶盘一起插入孔中。用胶带粘住右上角，以帮助在黑暗中定位板以进行成像。用半透明的柔性薄膜密封板，并用铝箔包裹板。在铝箔上写下绘图ID和板ID。将板放在黑暗的盒子或橱柜中至少30分钟，以放松非光化学淬火的前两个阶段。如果对非光化学淬灭的长期阶段感兴趣，请在成像前使用一小时的长时间暗潜伏期。通过在新鲜96孔板的每个角落放置一个叶盘并在中心放置一个叶盘，准备一个额外的假板，以便在分析过程中聚焦。像以前的板一样用鼻过滤器固定叶盘，密封板，并在室温和50%相对湿度的黑暗中孵育。打开成像仪并打开成像软件。单击“设置”，然后单击“协议”以打开一个窗口，用于在 PAM 实验协议中输入步骤。设置手稿中提供的机器的技术规格。将程序设置为从饱和脉冲开始，通过在名为“延迟之后”的框中输入20秒来测量暗适应光合作用的最大量子效率。单击“应用脉冲”框，然后在标题为“此次数”的框中输入 1。将脉冲 PPFD 设置为 6， 152，将脉冲长度设置为 800 毫秒，并选中“使用所有脉冲拍摄 F 素数和 FM 素数图像”框。选中“插入后”以向协议添加第二个步骤。在标题为“延迟之后”的框中输入 30 秒。然后，选择选项“更改光化”，并在标题为“光化 PPFD”的框中输入 50，将腔室的光强度设置为 50 PPFD。单击“插入时间后”向协议添加新步骤，并在标题为“延迟之后”的框中输入 150 秒。选择“应用脉冲”，然后在标题为“此次数”的框中输入 4，以便在光化光保持在 50 PPFD 时连续四次应用测量脉冲。根据手稿中提供的值调整每个步骤的延迟和光强度，然后将实验方案另存为所需位置的pcl文件。关闭灯并将假板面朝下放在样品台上，使叶子的轴表面朝上指向探测器，海绵朝下朝向平台。设置样品台高度，使叶盘高于仪器底座140毫米。单击连接/断开连接至成像仪相机和硬件相机图标以启动相机。单击焦点荧光符号（由底部的红色双面箭头图标和两条绿线表示），然后调整透镜和曝光以使板对焦。再次单击焦点荧光图标以关闭闪烁的指示灯。在黑暗中工作，用要分析的板替换假板。将其面朝下放置，外部用于定向右上角的胶带，然后单击地图图像相机图标。通过打开或关闭光圈来调整图像曝光，直到弹出窗口中的条形位于绿色区域中。每次调整光圈后，单击“重试”按钮，直到正确调整曝光并拍摄图像。右键单击图像，然后选择应用图像隔离以阻止背景信号。聚焦的叶区域将以灰色显示，背景以蓝色显示。通过右键单击图像，从修改图像下拉菜单中调整直方图和伽马水平，选择感兴趣的区域或像素以仅包含叶盘。右键单击图像，然后选择删除高切口和低切口以删除浅蓝色的高亮区域。右键单击图像，然后选择“删除杂散”以删除不接触最后三个其他像素的任何像素。影像上显示为孤立岛屿的任何区域都将单独进行分析，并包含在最终数据输出中。单击运行协议图标以启动程序。屏幕底部将出现一个计时器，通知您协议还剩下多长时间。等到协议完成。单击文件并另存为，将数据另存为 igr 文件。在启动另一个样品板之前，单击窗口右上角的红叉关闭窗口。要导出叶绿素荧光数据，请在成像软件中打开igr文件，然后单击“文件”，然后单击“导出到文件夹”以创建一个包含所有必要文件的新文件夹。对田间大豆非光化学猝灭的典型测量表明，当叶盘暴露于每秒50微摩尔的低光下时，在初始饱和闪光以确定Fv / Fm之后，非光化学淬火小于1。此外，在转移到每秒2， 000微摩尔的高光下时，非光化学淬火在15分钟后增加到最大值4。失败的测量显示，在转移到高光下时，非光化学淬火的增加很小。成像仪内的定位板以及明确定义的叶盘采样和电镀计划对于确保数据链接到正确的基因型至关重要。该协议可以量化环境变量（如干旱和脱落）对非光化学淬火的影响，或评估不同冠层水平的非光化学淬火，以完善作物模型。

Research

JoVE Journal

Biology

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

高通量使用自动染色体的物理图谱<em&gt;原位</em&gt;杂交

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

科研

生物学

RNA Secondary Structure Prediction Using High-throughput SHAPE

使用高通量形状的RNA二级结构预测

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Precise, High-throughput Analysis of Bacterial Growth

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

High-throughput Measurement of Gut Transit Time Using Larval Zebrafish

利用斑马鱼对肠道转运时间进行高通量测量

Zebrafish are used as alternative model organisms for drug safety testing. The gastrointestinal (GI) tract of zebrafish has genetic, neuronal, and ...

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

通过叶绿素荧光分析评估光呼吸突变体的光合效率

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the ...

Determination of Self-(In)compatibility and Inter-(In)compatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

Determination of Self-Incompatibility and Inter-Incompatibility Relationships in Citrus Using Manual Pollination, Microscopy, and S-Genotype Analyses

使用手动授粉、显微镜和 S 基因型分析测定柑橘中的自（内）相容性和相互（内）相容关系

Citrus uses S-RNase-based self-incompatibility to reject self-pollen and, therefore, requires nearby pollinizer trees for successful pollination and ...

https://cloudfront.jove.com/CDNSource/teasers/65056.jpg

在 Bamboo 中开发基因转化和编辑方法

In Planta Gene Expression and Gene Editing in Moso Bamboo Leaves

作者聚焦：推进竹叶基因编辑以获得可持续的塑料替代品 

在普兰塔 毛竹叶片基因表达与基因编辑

A novel in planta gene transformation method was developed for bamboo, which avoids the need for time-consuming and labor-intensive callus induction and ...