水杨酸(SA)是植物免疫的关键防御信号分子。植物SA的定量分析对于SA代谢途径及其生物学功能研究至关重要。利用高效液相色谱仪(HPLC)和液相-质谱联用仪(LC-MS)测定SA含量是目前常用方法, 但难以实现高通量测定。水稻(Oryza sativa)中SA合成代谢途径目前尚未完全解析, 高效筛选水稻SA相关突变体对于阐明其代谢途径具有重要意义。该文对已有基于SA生物传感菌株Acinetobacter sp. ADPWH_lux估算SA的分析方法进行了改良, 建立了水稻SA高通量估算方法, 简化了样品采集和提取过程, 省去样品称重、组织研磨及离心等耗时步骤, 整个操作流程便捷且高效。同时, 利用已报道的水稻SA代谢相关遗传材料验证了该方法的可行性, 并使用该方法筛选了钴-60诱变的水稻突变体库, 获得一批水稻SA含量发生显著变化的突变体, 采用HPLC法对突变体内源SA进行了验证。该方法可用于SA代谢突变体的遗传筛选及其代谢相关酶鉴定, 对水稻等作物的SA代谢及生物学功能研究具有重要的应用价值。

INTRODUCTION Salicylic acid (SA) plays an important role in the plant immune system. The quantitative analysis of SA in plants is fundamental to studying SA metabolism and its biological functions. Although high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) are widely used for SA determination, their low throughput limits their suitability for large-scale analysis. However, the SA biosynthetic pathway in rice is not well understood, highlighting the need for efficient methods to screen SA-related mutants and elucidate SA metabolic pathways.

RATIONALECurrent methods for measuring endogenous SA levels, such as HPLC and LC/MS, involve labor-intensive sample preparation, making them unsuitable for high-throughput analysis. While a lux gene-based SA biosensor has been successfully used in tobacco and Arabidopsis, a reliable and efficient method for SA detection in rice remains unavailable. To address this problem, we optimized sample processing and operational workflows to enable high- throughput SA quantification in rice plants.

RESULTS We developed a streamlined, high-throughput method for SA quantification in rice, eliminating time-consuming steps such as sample weighing, tissue grinding, and centrifugation. This approach significantly simplifies the process while maintaining efficiency and accuracy. We validated the method’s feasibility using published rice SA metabolic mutants. We then applied it to screen a Cobalt-60 induced rice mutant library, identifying mutants with altered SA metabolism. Endogenous SA levels in these mutants were confirmed using HPLC. The results demonstrate the method’s effectiveness in screening SA-related metabolic mutants, providing a valuable tool for studying SA metabolism and its roles in rice and other crops. The method was validated using known SA genetic materials. SA content-altered mutants were successfully isolated for further research.

CONCLUSION This study establishes a rapid and cost-effective method for measuring SA content in rice tissues using the SA biosensor Acinetobacter sp. ADPWH_lux. Given the pivotal role of SA in plant defense, our method adopts streamlined sampling process, requiring only leaf clipping and boiling in LB medium, and dramatically reduces the time and effort associated with tissue collection and processing. This high-throughput approach is well-suited for large-scale screening of greenhouse-grown or hydroponic plants, providing a powerful platform for advancing research on SA metabolism and its biological functions in crops.

Modified manipulating process for high-throughput determination of salicylic acid (SA) content using SA biosensor Acinetobacter sp. ADPWH_lux strain in rice