Department of Microbiology and Cell Science, University of Florida, P.O. Box 110700, Gainesville, FL, 32611, USA.
Plant Methods. 2010 Sep 23;6:21. doi: 10.1186/1746-4811-6-21.
Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor Acinetobacter sp. ADPWH_lux-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.
On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, npr1 (nonexpressor of pathogenesis-related genes), and sid2 (SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six npr1 suppressors that accumulated lower levels of SA than npr1 upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified eds5 mutant. The other four are more susceptible than npr1 to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 and their identity merits further investigation.
The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor Acinetobacter sp. ADPWH_lux-based SA estimation, and allowed the screening for npr1 suppressors that accumulated less SA than npr1 after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.
水杨酸（SA）是植物中抵抗生物营养病原体的关键防御信号分子。植物中 SA 水平的定量分析对于解析 SA 介导的免疫反应至关重要。尽管高效液相色谱法（HPLC）和气相色谱-质谱法（GC/MS）常用于测定 SA 浓度，但这些方法昂贵且耗时。我们最近描述了一种基于 Acinetobacter sp. ADPWH_lux 的快速细菌生物传感器定量 SA 的方法，该方法能够进行高通量分析。在本研究中，我们描述了一种用于快速样品制备的改进方法，并提出了一种用于分离 SA 代谢突变体的高通量策略。
基于之前描述的基于生物传感器的方法，我们简化了组织收集和 SA 提取步骤。收集叶片圆盘并在 LB 中煮沸，然后用生物传感器测量释放的 SA。避免了称重样品、研磨组织和离心等耗时步骤。直接煮沸方案检测到与以前描述的基于生物传感器的方法和基于 HPLC 的方法相似的野生型、npr1（无致病性相关基因表达）和 sid2（SA 诱导缺陷）植物之间的 SA 水平差异，证明了本研究中提出的方案的有效性。我们将该方案改编为高通量格式，并鉴定出六个 npr1 抑制剂，它们在病原体感染时比 npr1 积累的 SA 水平更低。其中两个抑制剂被鉴定为以前鉴定的 eds5 突变体的等位基因。另外四个比 npr1 对细菌病原体丁香假单胞菌 pv. maculicola ES4326 更敏感，它们的身份值得进一步研究。
通过直接煮沸叶片圆盘进行快速的 SA 提取方法进一步降低了基于 Acinetobacter sp. ADPWH_lux 的生物传感器 SA 估计所需的成本和时间，并且允许以高通量方式筛选在病原体感染后比 npr1 积累更少 SA 的 npr1 抑制剂。这种高效的 SA 估计方案可应用于 SA 代谢突变体的遗传筛选和参与 SA 代谢的酶的特征分析。本研究中分离的突变体可能有助于确定 SA 相关信号通路中的新成分。
