这项研究是由耶鲁大学气候变化与能源研究所和美国国家科学基金会的资金支持。

1。应力和压力条件下饲养草蜢<ol><li>使用0.5米2圆形的，封闭的生物群落，，防止移民或移民的动物物种（ 图1）。建设生物群落使用2.4米长，1.5米高的¼“目铝围栏脚手架。量2.5米长1.75米高的铝窗纱折叠的顶部和底部的击剑击剑和，沿折叠装订在一起。加入击剑结束形成一个封闭的圆，然后装订重叠筛选，共同创造一个密封的窗口，设置围隔领域的土壤中挖一个10厘米深，4厘米宽的沟基地周围的mesocosom，沉围隔成沟槽，然后夯实草皮周围的沟槽。短纤维窗纱一块圆形的围隔顶部的凹陷部分的围隔。 </li><li>阵列生物群落在复制的配对实验desig该字段中的n。剧情地点应选择相匹配的植物种类的身份和植物相对盖。笼10厘米下沉到地面的情节现场。 </li><li>用扫网，收集早期（第2 次 ）龄蝗虫若虫和库存的生物群落在自然领域密度。 </li><li>用扫网，捕捉个体的主导坐，等待狩猎蜘蛛捕食物种（而不是web织）。胶具有快干水泥解耦风险的影响，从实际生存选择有利于个人更好的能力来回避蜘蛛捕食蝗虫与关蜘蛛的螯肢（口器用来制伏猎物）。股票的蜘蛛在现场密度每对围隔。这将是应力处理。没有蜘蛛的生态系统中的压力免费治疗。 </li><li>允许蝗虫若虫发展到晚期（4 日和5 日 ）龄阶段。收集的所有个人的笼子里，RAndomly将个人从每个网箱后的三个试验组之一：（1）验证的生理应激状态;（2）验证在体内元素的化学计量学的转变;（3）微生物分解。 </li></ol> 2。验证草蜢应力状态<ol><li>测量蝗虫标准代谢率（SMR），二氧化碳排放量的速度（ <img src="/files/ftp_upload/50061/50061eq1.jpg" fo:src="/files/ftp_upload/50061/50061eq1highres.jpg"/> ）在一个incurrent流通过呼吸测量系统与200毫升/分钟的空气流速。通过干燥剂通过流动的空气中除去水蒸气。 </li><li>食品剥夺16小时（水应该是可用的），衡量个人蝗虫（±0.1毫克），并将其放置在透明50毫升（9.2厘米长x 2.0厘米厚）呼吸室，并允许他们回收处理至少测量前10分钟开始。 </li><li>在恒定的环境温度平均<html>内的呼吸测量室的温度（温度±1％标准误差变化），分析呼吸消耗空气使用红外线的CO 2分析仪（ 例如比特的S151-1 ppm分辨率）。测量的平均值最小的稳态<img src="/files/ftp_upload/50061/50061eq1.jpg" fo:src="/files/ftp_upload/50061/50061eq1highres.jpg"/>为10分钟。 </li><li>分析仪提供了分数CO 2浓度（每百万），但应填报为SMR的速度，因此，我们必须转 ​​变录音<img src="/files/ftp_upload/50061/50061eq1.jpg" fo:src="/files/ftp_upload/50061/50061eq1highres.jpg"/> = FR（ <img src="/files/ftp_upload/50061/50061eq2.jpg" fo:src="/files/ftp_upload/50061/50061eq2highres.jpg"/> - <img src="/files/ftp_upload/50061/50061eq3.jpg" fo:src="/files/ftp_upload/50061/50061eq3highres.jpg"/> ）/ {1 - <img src="/files/ftp_upload/50061/50061eq2.jpg" fo:src="/files/ftp_upload/50061/50061eq2highres.jpg"/> [1 - （1/RQ）]}，其中iles/ftp_upload/50061/50061eq3.jpg“佛：保证”/ files/ftp_upload/50061/50061eq3highres.jpg“/&gt; = incurrent的小数的CO 2浓度; <img src="/files/ftp_upload/50061/50061eq2.jpg" fo:src="/files/ftp_upload/50061/50061eq2highres.jpg"/> = excurrent分数的CO 2浓度; FR =流速（毫升分钟-1）; RQ =呼吸商，假定等于0.85草食动物。 </li></ol> 3。验证移位体元素化学计量学<ol><li>评估碳：氮（C：N）含量的蝗虫从生物群落收集的样本。 </li><li>减少变异在C：N，由于近期出现的食品消费在解剖显微镜下删除蝗虫肠道内容。 </li><li>冷冻干燥空的肠道和身体48小时，然后磨个人的屠体和内脏均匀的粉末。 </li><li>测量C：N含量的粉末用CNH自动分析仪。 </li></ol> 4。微生物分解<ol><li>地点复制对聚氯乙烯塑料铤（15.4厘米直径，插入〜4公分到土壤中）在现场（ 图3C）。通过在土壤表面的削波内删除所有的植被。这些项圈用于分解的措施。此外，建立一套整个现场的PVC衣领作为13 C自然丰度的控制（见下文），既不是蝗虫，也不是草枯落物中添加。 </li><li>衣领每对中的添加2条完整，冻干尸体，蝗虫（记录的生物量增加），如上述使用领域笼饲养与捕食风险。每对中的其他领加2条完整冻干尸体饲养，没有捕食的风险。 </li><li>覆盖PVC铤的画面，，防止蝗虫去除拾荒者的情节，让蝗虫的尸体分解为40天。 </li><li>虽然尸体被分解，标签草里施泰特有13℃。 <ol><li>构建一个明确的有机玻璃室（60厘米×60厘米×1.5米）的入口和出口阀（ 图3B）。 </li><li>水槽的正方形的60厘米×60厘米木框与涂覆用硅润滑脂5厘米的到地面（ 图3B）中的橡胶密封件。 </li><li>滑动室的木制框架的顶部，使得腔室成为密封的橡胶（ 图3B）。 </li><li>室进气口连接，含有99％（原子）13 CO 2的压缩气体钢瓶。室内的植物都将被贴上13 C，CO 2浓度保持在环境水平（因为提升的浓度改变植物中的碳分区）。环境水平仅由脉冲标记CO 2短的时间内保持。此外，室内的温度进行监控室被删除，如果温度达到5℃;波夫环境。 </li><li>一个星期后的标签，比较δ13 C与自然丰度值相同的草种，使用一个热DeltaPlus同位素比质谱仪（赛，圣何塞，CA，USA）的随机抽样收集垃圾的草。 </li></ol></li><li>经过40天，加10克风干13 C标记的草地蝗虫尸体此前被修订，各领垃圾。 </li><li>监测的草地凋落物的原位在75天的上限各领和跟踪土壤呼吸和呼吸CO 2 13的矿化率。这是通过使用一个流室技术，实时监测气体样品各领8分钟，每个使用光腔衰荡光谱（CRDS Picarro公司，圣克拉拉，CA，USA;型号：G1101 -i）的。 CRDS可同时跟踪总额和δ13 C土壤呼吸。 </li><li>估计13 C标记的草-枯落物利用同位素混合方程对土壤呼吸的贡献。草枯落物来源的CO 2量的计算方法如下：Ç 草枯落物而得 = C 总量 ×（δ13 C 呼吸 - δ13 C nat.abn）/（δ13 C 草-枯落物 - δ13 C NAT 。荷兰 ），其中C 总的总额是在给定的测量呼吸的C，δ13 C 呼吸的δ13 C-C呼吸消耗的衣领修订标记草枯枝落叶，δ13 C nat.abn。是的平均δ13 C呼吸消耗ç在三个自然丰度铤（ 即那些没有修订，乱抛垃圾）和δ13 C 基层枯枝落叶的草地上乱抛垃圾的δ13 C的共同llars。 </li><li>同时监测土壤温度和水分在整个实验中使用的手持式探头纠正由于温度和湿度的差异，土壤呼吸的差异。 </li><li>虽然用于现场使用，光腔衰荡光谱仪（Picarro公司，圣克拉拉，CA，USA;型号：G1101-I）的读数是敏感的运动。因此，所有的地块含有PVC衣领竖立基地测量站，并与PVC管的长度，将仪器连接到衣领。 </li></ol>

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我们什么都没有透露。

这里介绍的方法的顺序应该让系统的压力测量在地上食物网的种类包括素土微生物群落的方式，导致变更后凋落物的分解。是理想的，因为完整的食物网可以在空间上受限制和包含在生物群落为研究，包括节肢动物消费者和草本植物的生态系统的方法。 空间变异可能存在由于梯度背景土壤水分，土壤温度，植物养分含量等的研究设计，让一到的阵列mescosms和PVC衣领挡住沿空间的环境梯度，从而账户等环境变化进行分析时的动画效果。 虽然用于现场使用，光腔衰荡光谱仪（Picarro公司，圣克拉拉，CA，USA;型号：G1101-I）的读数本身nsitive运动。因此，所有的地块含有PVC衣领竖立基地测量站，并与PVC管的长度，将仪器连接到衣领。 土壤枯枝落叶分解历来被测量封闭已知的垃圾数量为玻璃纤维网袋，袋存放在该领域在土壤表面，并定期重新测量袋量化凋落物消失率（分解）。这种方法的局限性是，一个是无法追踪的命运的分解物或确定CO 2矿化的土壤改良剂（添加枯枝落叶）的背景土壤CO 2矿化的贡献。这里介绍使用标记的CO 2示踪方法有助于缓解这一后勤约束。 生态系统生态学和生物地球化学的操作下工作的范例，因为吃剩的植物-枯落物包括多数碎屑，地下生态系统过程的影响只是轻微的生物质投入，从更高的营养水平在地上的食物网，如草食动物自己6。然而，有越来越多的证据表明，可以有更高的营养水平的物种在生态系统地下过程产生深远的影响1,4,5。这里介绍的方法，站在更高的营养水平，以提高定量的贡献，无论是通过生物 ​​质直接从尸体沉积（ 如：12，13）或排泄和排遗作用（ 如 14，15）或间接通过改变植物群落组成（ 例如 9 ）对生态系统的养分循环。这种量化有助于揭示动物的机制，控制生态系统动态协同努力的一部分，以加强和修改当前的工作范式的生物控制生态系统的功能。

<table border="1"><tbody><tr><td> 的试剂或设备的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>腔衰荡光谱仪</td><td> Picarro公司，圣克拉拉，CA，USA </td><td>型号＃G1101-I </td><td></td></tr><tr><td> CO2呼吸</td><td>量子比特的系统，金士顿，ON，加拿大</td><td>型号＃S151 </td><td></td></tr><tr><td> 13C </td><td> Sigma-Aldrich公司</td><td> 372382 </td><td></td></tr><tr><td>分光光度计</td><td>热，美国加利福尼亚州圣何塞</td><td>型号：增量V +同位素比质谱仪</td><td></td></tr></tbody></table>

linking predation risk, herbivore physiological stress and microbial decomposition of plant litter

碎屑进入土壤的数量和质量决定由微生物群落以及作为再循环率的氮（N），碳（C）封存1,2的分解速率。包括大多数植物凋落物碎屑，因此它假定分解生物质输入动物，如草食动物和肉食动物4,5只轻微影响。然而，的食肉动物可能影响微生物分解的植物残体通过链的互动中，他们的草食动物的猎物捕食风险的生理改变，反过来又改变了土壤微生物的功能，当草食动物尸体分解。草食动物的生理应激反应捕食的风险，可以改变C：N元素组成的草食动物生物量7,8,9，因为捕食风险增加草食动物基础的能源需求的压力，在营养系统的力量有限草食动物s到他们的消费转移，从N-丰富的资源，以支持经济增长和繁殖，C丰富的碳水化合物资源，以支持提高新陈代谢6。食草动物都将多余的营养物质的能力有限，所以强调草食动物排泄，N为他们增加碳水化合物-C消耗7。最终，捕食所强调的捕食风险，提高其机身C：N比7,10，质量较差的资源，土壤微生物池可能是由于较低的可用性不稳定的Ň微生物酶生产6。因此，压力草食动物的尸体分解微生物群落的功能，减少后续能力的微生物分解，植物凋落物6,10,11一个启动效应。 我们提出的方法来评估捕食风险之间的联系和凋落物分解土壤中的微生物。我们描述了如何产生应力草食动物捕食风险;测量确保这些应激反应，和测量的后果微生物分解。我们使用的见解，从模型草原生态系统，包括狩猎蜘蛛，捕食者（Pisuarina MIRA），占主导地位的的蚂蚱的食草动物（Melanoplus femurrubrum），以及各种草和杂类草植物9。

图2给出了一个例子情节的蚂蚱应力和组织应力条件下的标准代谢率的。由于个人蝗虫的身体质量差异，而事实上，代谢率与体重变化，情节应当出示代谢率的关系蚱蜢的身体质量。不同的治疗方法并行的趋势表明，代谢率的上升都是强调个人作为一个恒定的标准代谢率的倍数（ 即有没有身体质量×代谢率相互作用）。 蝈蝈体C和N元素含量的风险和无风险的条件列于表1。值得注意的是，有一个非常小的（4％）治疗之间的区别在机身C：N比。然而，这些微小的差别可以转化为差异大草地凋落物的分解的土壤微生物池（ 图3）。 </p&gt; 草枯枝落叶PVC的衣领以前修订与压力或无压力的蝗虫会导致不同程度的凋落物的分解，反映在曲线描述从土壤中微生物呼吸（ 图3）由于累积CO 2释放。应监测实验，直到累积曲线开始饱和。 <table border="1"><tbody><tr><td></td><td> 应力 </td><td> 强调自由 </td></tr><tr><td>碳（％） </td><td> 48.44±0.32 </td><td> 44.73±0.46 </td></tr><tr><td>氮（％） </td><td> 12.11±0.08 </td><td> 11.62±0.12 </td></tr><tr><td>碳：氮</td><td> 4.00±0.03 </td><td> 3.85±0.04 </td></tr></tbody></table> 表1的蚂蚱食草动物车的化学成分比较。casses从条件，使他们面对捕食风险（压力），并在捕食风险是不存在的（压力）。这些值是平均值±1个标准误差。 <img src="/files/ftp_upload/50061/50061fig1.jpg" alt="图1"/> 图1。插图的生态系统中用于试验总体方案的的实验评估风险的影响，凋落物分解的设计。 <img src="/files/ftp_upload/50061/50061fig2.jpg" alt="图2"/> 图2：情节标准代谢率的草食动物，草食动物的身体质量。的数据是根据实验性治疗分为两大类：蝗虫从食肉动物（捕食）产生应力，生物群落的生物群落，没有捕食者（控制），因此没有引起应力。数据D. HalwenaOJ施密茨2010年出版。 <img src="/files/ftp_upload/50061/50061fig3.jpg" alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/50061/50061fig3highres.jpg"/> 图3描述累计CO 2释放的微生物池，而PVC衣领分解实验草凋落物输入的曲线。绘制的值的平均值±1个标准误差。该图显示了引物，土壤与压力的蝗虫尸体（捕食者）低19％（ANOVA F 1,6 = 9.06，P &lt;0.05），无压力的蝗虫尸体（对照组）引物与土壤的植物凋落物的分解率比。插图显示设备在该领域的PVC领。图转载从Hawlena 等。的 <a href="https://www.jove.com/files/ftp_upload/50061/50061fig3large.jpg" target="_blank">依次点击这里查看大图</a> 。

Watch this Scientific Journal Video about Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter at JoVE.com

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

我们提出的方法，以评估如何捕食风险加剧的压力导致饮食结构的改变，以满足需求，可以改变化学品质的草食动物的猎物，以及如何从这些压力草食动物尸体的分解减慢随后的植物凋落物分解土壤中的微生物。

链接捕食风险，草食动物生理应激和植物凋落物的微生物分解

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

linking-predation-risk-herbivore-physiological-stress-microbial

Research

JoVE Journal

Biology

12.9K 次观看.  Yale University. 我们提出的方法，以评估如何捕食风险加剧的压力导致饮食结构的改变，以满足需求，可以改变化学品质的草食动物的猎物，以及如何从这些压力草食动物尸体的分解减慢随后的植物凋落物分解土壤中的微生物。

视频: Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

Testing the Physiological Barriers to Viral Transmission in Aphids Using Microinjection

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When an uninfected aphid feeds on an infected plant it contracts the virus through the plant phloem. Once ingested, the virus must pass from the insect gut to the hemolymph (the insect  blood ) and then must pass through the salivary gland, in order to be transmitted back to a new plant. An aphid may take up different viruses when munching on a plant, however only a small fraction will pass through the gut and salivary gland, the two main barriers for transmission to infect more plants. In the lab, we use physalis plants to study PLRV transmission. In this host, symptoms are characterized by stunting and interveinal chlorosis (yellowing of the leaves between the veins with the veins remaining green). The video that we present demonstrates a method for performing aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut is preventing viral transmission.
The video that we present demonstrates a method for performing Aphid microinjection on insects that do not vector PLVR viruses and tests whether the gut or salivary gland is preventing viral transmission.

Aphids are effective transmitters of plant viruses. Aphid microinjection of virus, the procedure we will show you today, is a technique allowing researchers to inject virus directly into the hemocoel of the aphid, bypassing the gut, one of the 2 major barriers for virus transmission in a circulative manner. The same technique is also used to inject dsRNA for RNAi.

测试使用微量注射蚜虫病毒传播的生理障碍

Potato loafroll virus (PLRV), from the family Luteoviridae infects solanaceous plants. It is transmitted by aphids, primarily, the green peach aphid. When ...

科研

生物学

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

植物激素在应激反应监测

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

为控制植物组织分解好氧菌的琼脂块缩影

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2. These HIPVs are mainly regulated by the defensive plant hormone jasmonic acid (JA) and its volatile derivative methyl jasmonate (MeJA)3,4,5. Over the past 3 decades researchers have documented that HIPVs can repel or attract herbivores, attract the natural enemies of herbivores, and in some cases they can induce or prime plant defenses prior to herbivore attack. In a recent paper6, I reported that feeding by gypsy moth caterpillars, exogenous MeJA application, and mechanical damage induce the emissions of volatiles from blueberry plants, albeit differently. In addition, blueberry branches respond to HIPVs emitted from neighboring branches of the same plant by increasing the levels of JA and resistance to herbivores (i.e., direct plant defenses), and by priming volatile emissions (i.e., indirect plant defenses). Similar findings have been reported recently for sagebrush7, poplar8, and lima beans9..
Here, I describe a push-pull method for collecting blueberry volatiles induced by herbivore (gypsy moth) feeding, exogenous MeJA application, and mechanical damage. The volatile collection unit consists of a 4 L volatile collection chamber, a 2-piece guillotine, an air delivery system that purifies incoming air, and a vacuum system connected to a trap filled with Super-Q adsorbent to collect volatiles5,6,10. Volatiles collected in Super-Q traps are eluted with dichloromethane and then separated and quantified using Gas Chromatography (GC). This volatile collection method was used n my study6 to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants. These methods are described here. Briefly, undamaged blueberry branches are exposed to HIPVs from neighboring branches within the same plant. Using the same techniques described above, volatiles emitted from branches after exposure to HIPVs are collected and analyzed.

A push-pull method for collecting plant volatiles is described. The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage. This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

蓝莓虫害诱导挥发物和植物内信令

Herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack1,2.  These HIPVs are mainly regulated by the defensive ...

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti

Using two methods to estimate gene expression in the major gustatory appendages of Aedes aegypti, we have identified the set of genes putatively underlying the neuronal responses to bitter and repulsive compounds, as determined by electrophysiological examination.

生理录音和黄热病蚊子的味觉附属物RNA测序<em&gt;埃及伊蚊</em

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

结合透明和荧光显微镜，可视化基因表达的变化和对芸苔疟原虫的生理反应

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

Scalable, Flexible, and Cost-Effective Seedling Grafting

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs.

可扩展、灵活且经济高效的育苗嫁接

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage ...