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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
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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