DataSheet_1_Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory.pdf

Feeding by chewing insects induces chemical defenses in plants that are regulated by the jasmonic acid (JA) pathway. Jasmonates are usually quantified by liquid chromatography–mass spectrometry (LC-MS) analysis of precursors and products in the biosynthetic pathway or inferred from the extraction and expression of genes known to respond to elevated levels of JA. Both approaches are costly and time consuming. To address these limitations, we developed a rapid reporter for the synthesis of JA based on the OPR3promoter:YFP-PTS1. Yellow fluorescent protein (YFP) fluorescence was increased by mechanical wounding and methyl jasmonate (MeJA) treatment and by caterpillar feeding. To develop an optimal sampling time for a quantitative bioassay, OPR3promoter:YFP-PTS1 plants were sampled at 1, 2, 3, and 24 h after treatment with 115 µM MeJA. The first increase in YFP fluorescence was detected at 2 h and remained elevated 3 and 24 h later; as a result, 3 h was chosen as the sampling time for a quantitative bioassay of jasmonate response to insect attack. Feeding by Pieris rapae caterpillars induced a 1.8-fold increase in YFP fluorescence, consistent with the known induction of JA production by this insect. We also assessed the utility of this reporter in studies of plant responses to caterpillar feeding vibrations, which are known to potentiate the JA-dependent production of chemical defenses. Pretreatment with feeding vibrations increased expression of the OPR3promoter:YFP-PTS1 in response to 14 µM MeJA. Feeding vibrations did not potentiate responses at higher MeJA concentrations, suggesting that potentiating effects of prior treatments can only be detected when plants are below a response threshold to the elicitor. The expression of OPR3 does not indicate levels of specific downstream jasmonates and quantification of specific jasmonates still requires detailed analysis by LC-MS. However, OPR3 expression does provide a rapid and inexpensive way to screen large numbers of plants for the involvement of jasmonate signaling in their response to a wide variety of treatments, and to study the induction and expression of AtOPR3.

咀嚼式昆虫取食会诱导植物产生由茉莉酸（jasmonic acid, JA）通路调控的化学防御反应。茉莉酸类化合物通常通过液相色谱-质谱联用法（liquid chromatography–mass spectrometry, LC-MS）分析其生物合成通路中的前体与产物进行定量，或是通过提取并检测已知响应JA水平升高的基因的表达量来间接推断。这两种方法均存在成本高昂、耗时较长的缺陷。为解决上述局限，我们基于OPR3启动子:YFP-PTS1构建了一种用于检测JA合成的快速报告系统。黄色荧光蛋白（yellow fluorescent protein, YFP）的荧光信号可通过机械损伤、茉莉酸甲酯（methyl jasmonate, MeJA）处理以及昆虫幼虫取食而增强。为确定定量生物检测的最优采样时间，我们对经115 μM MeJA处理后的OPR3启动子:YFP-PTS1植株分别在1、2、3和24小时进行采样。YFP荧光信号在处理后2小时首次出现上升，并在3小时和24小时时仍维持较高水平；因此，我们选定3小时作为定量检测昆虫取食诱导JA响应的采样时间。菜青虫（Pieris rapae）幼虫取食可使YFP荧光信号提升1.8倍，这与该昆虫诱导JA合成的已有研究结果一致。我们还评估了该报告系统在植物响应幼虫取食振动相关研究中的应用价值——已知此类振动可增强依赖JA的化学防御产物合成。经取食振动预处理后，植株在响应14 μM MeJA时OPR3启动子:YFP-PTS1的表达量有所提升。但当MeJA浓度较高时，取食振动并未增强上述响应，这表明先前处理的增强效应仅在植株对激发子（elicitor）的响应处于阈值以下时才可被检测到。OPR3的表达量无法反映特定下游茉莉酸类化合物的水平，若要定量特定茉莉酸类化合物，仍需通过LC-MS进行详细分析。然而，OPR3的表达确实提供了一种快速且低成本的方法，可用于筛选大量植株以检测茉莉酸信号通路是否参与其对多种处理的响应，同时也可用于研究AtOPR3的诱导与表达模式。