水稻水杨酸代谢突变体高通量筛选方法的建立与应用

doi:10.11983/CBB24148

摘要/Abstract

摘要： 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。高效液相色谱仪(HPLC)和液相-质谱联用仪(LC/MS)是测定SA含量的常用方法, 但难以实现高通量测定。水稻(Oryza sativa)合成代谢途径尚未完全解析, 高效筛选SA相关突变体对于解析其代谢途径具有重要意义。该文针对已有的基于SA生物传感菌株Acinetobacter sp. ADPWH_lux估算SA的分析方法进行改良, 建立了水稻SA高通量估算方法, 对样品采集以及提取过程进行了简化, 省去了样品称重、组织研磨和离心等耗时步骤, 整个操作流程便捷高效。我们利用已报道的水稻SA代谢相关遗传材料证明了该方法的可行性, 同时用该方法筛选钴-60诱变的水稻突变体库, 获得了一批水稻SA含量发生显著变化的突变体, 并利用HPLC法对突变体内源SA进行验证。该方法可用于SA代谢突变体的遗传筛选和SA代谢相关酶的鉴定, 对于水稻等作物的SA代谢及生物学功能研究具有重要应用价值。

关键词: 水杨酸测定, 生物传感器, 高通量方法, 水杨酸代谢突变体, 水稻

Abstract: Salicylic acid (SA) plays essential roles in plant immunity. Quantitative analysis of SA in plants is essential for studying SA metabolism and biological function. Although high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC/MS) are widely used for SA determination, but it is difficult to achieve high throughput determination. However, SA synthesis pathway in rice has not yet to be fully elucidated, efficient screening method of SA-related mutants is of great significance for the analysis of the metabolic pathway. In this paper, we improved the analytical method for quantifying SA based on biosensor Acinetobacter sp.ADPWH_lux, and established a rapid high-throughput method for SA quantification in rice with simplifying the sample collection and extraction process. It avoids the time-consuming steps such as sample weighing, tissue grinding and centrifugation, and the entire process is convenient and efficient. We demonstrated the method's feasibility using the published rice SA metabolism mutants. Then, we screened some rice mutants from Co-60 mutant library with high or low SA metabolism using this method and confirmed the source SA level of the mutants by HPLC. Our results showed that this method could be used to screen SA related metabolic mutants, and has important application potential for studying the role of SA metabolism and biological could function in rice and other crops.

叶灿, 姚林波, 金莹, 高蓉, 谭琪, 李旭映, 张艳军, 陈析丰, 马伯军, 章薇, 张可伟. 水稻水杨酸代谢突变体高通量筛选方法的建立与应用. 植物学报, DOI: 10.11983/CBB24148.

Can Ye, Linbo Yao, Ying Jin, Rong Gao, Qi Tan, Xuying Li, Yanjun Zhang, Xifeng Chen, Bojun Ma, Wei Zhang, Kewei Zhang. Establishment and Application of a High-throughput Screening Method for Salicylic Acid Metabolic Mutants in Rice. Chinese Bulletin of Botany, DOI: 10.11983/CBB24148.

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参考文献

[1]李超, 周琳, 张永军, 宋福平, 张杰(2009).离子环境对Acinetobacter sp ADP1的salR.基因活性的影响.生物技术通报, :57-63. [2]Ahmad P, Prasad M(2012).Abiotic stress responses in plants.Springer, :235-251. [3]Dempsey DA, Vlot AC, Wildermuth MC, Klessig DF(2011).Salicylic acid biosynthesis and metabolism. .Arabidopsis Book, 9:e0156-. [4]Ding P, Ding Y(2020).Stories of salicylic acid: a plant defense hormone.Trends Plant Sci, 25:549-565. [5]Gaffney T, Friedrich L, Vernooij B, Negrotto D, Nye G, Uknes S, Ward E, Kessmann H, Ryals J(1993).Requirement of salicylic acid for the induction of systemic acquired resistance.Science, 261:754-756. [6]Garcion C, Lohmann A, Lamodière E, Catinot J, Buchala A, Doermann P, Métraux JP(2008).Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of arabidopsis.Plant Physiol, 147:1279-1287. [7]Huang WE, Huang L, Preston GM, Naylor M, Carr JP, Li Y, Singer AC, Whiteley AS, Wang H(2006).Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1. .Plant J, 46:1073-1083. [8]Huang WE, Wang H, Zheng H, Huang L, Singer AC, Thompson I, Whiteley AS(2005).Chromosomally located gene fusions constructed in Acinetobacter sp.ADP1 for the detection of salicylate..Environ Microbiol, 7:1339-1348. [9]Jiang G, Yin D, Shi Y, Zhou Z, Li C, Liu P, Jia Y, Wang Y, Liu Z, Yu M, Wu X, Zhai W, Zhu L (2020).OsNPR3.3-dependent salicylic acid signaling is involved in recessive gene xa5-mediated immunity to rice bacterial blight. .Sci Rep , 10:6313-6313. [10]Koo YM, Heo AY, Choi HW(2020).Salicylic acid as a safe plant protector and growth regulator.The Plant Pathol J, 36:1-10. [11]Kouzai Y, Kimura M, Watanabe M, Kusunoki K, Osaka D, Suzuki T, Matsui H, Yamamoto M, Ichinose Y, Toyoda K, Matsuura T, Mori IC, Hirayama T, Minami E, Nishizawa Y, Inoue K, Onda Y, Mochida K, Noutoshi Y(2018).Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani,a necrotrophic fungal agent of sheath blight,in rice and Brachypodium distachyon.New Phytol, 217:771-783. [12]Liu S, Wu Y, Fang C, Cui Y, Jiang N, Wang H(2017).Simultaneous determination of 19 plant growth regulator residues in plant-originated foods by QuEChERS and stable isotope dilution-ultra performance liquid chromatography-mass spectrometry.Anal Sci, 33:1047-1052. [13]Malamy J, Carr JP, Klessig DF, Raskin I(1990).Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection.Science, 250:1002-1004. [14]Marek G, Carver R, Ding Y, Sathyanarayan D, Zhang X, Mou Z(2010).A high-throughput method for isolation of salicylic acid metabolic mutants..Plant Methods, :21-21. [15]Meighen EA(1993).Bacterial bioluminescence: organization,regulation,and application of the lux genes.FASEB J, 7:1016-1022. [16]Métraux JP, Signer H, Ryals J, Ward E, Wyss-Benz M, Gaudin J, Raschdorf K, Schmid E, Blum W, Inverardi B(1990).Increase in salicylic acid at the onset of systemic acquired resistance in cucumber.Science, 250:1004-1006. [17]Peng Y, Yang J, Li X, Zhang Y(2021).Salicylic acid: biosynthesis and signaling.Annu Rev Plant Biol, 72:761-791. [18]Rasmussen JB, Hammerschmidt R, Zook MN(1991).Systemic induction of salicylic acid accumulation in cucumber after inoculation with Pseudomonas syringae pv syringae.Plant Physiol, 97:1342-1347. [19]Rekhter D, Ludke D, Ding Y, Feussner K, Zienkiewicz K, Lipka V, Wiermer M, Zhang Y, Feussner I(2019).Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid.Science, 365:498-502. [20]Rivas-San Vicente M, Plasencia J(2011).Salicylic acid beyond defence: its role in plant growth and development.J Exp Bot, 62:3321-3338. [21]Saleem M, Fariduddin Q, Castroverde CDM(2021).Salicylic acid: a key regulator of redox signalling and plant immunity.Plant Physiol Biochem, 168:381-397. [22]Sanders IO, Smith AR, Hall MA(1989).The measurement of ethylene binding and metabolism in plant tissue.Planta, 179:97-103. [23]Shields A, Shivnauth V, Castroverde CDM(2022).Salicylic acid and N-hydroxypipecolic acid at the fulcrum of the plant immunity-growth equilibrium.. Front Plant Sci , 13:841688-. [24]Shimono M, Sugano S, Nakayama A, Jiang CJ, Ono K, Toki S, Takatsuji H(2007).Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance.Plant Cell, 19:2064-2076. [25]Silverman P, Seskar M, Kanter D, Schweizer P, Metraux JP, Raskin I(1995).Salicylic acid in rice (biosynthesis,conjugation,and possible role).Plant Physiol, 108:633-639. [26]Sutcharitchan C, Miao S, Li W, Liu J, Zhou H, Ma Y, Ji S, Cui Y(2020).High performance liquid chromatography-tandem mass spectrometry method for residue determination of 39 plant growth regulators in root and rhizome Chinese herbs. .Food Chem, 322:126766-. [27]Van Butselaar T, Van Den Ackerveken G(2020).Salicylic acid steers the growth-immunity tradeoff.Trends Plant Sci, 25:566-576. [28]Waadt R, Seller CA, Hsu PK, Takahashi Y, Munemasa S, Schroeder JI(2022).Plant hormone regulation of abiotic stress responses.Nat Rev Mol Cell Biol, 23:680-694. [29]Wang Y, Jin G, Song S, Jin Y, Wang X, Yang S, Shen X, Gan Y, Wang Y, Li R, Liu JX, Hu J, Pan R(2024).A peroxisomal cinnamate: CoA ligase-dependent phytohormone metabolic cascade in submerged rice germination.Dev Cell, 59:1363-1378. [30]Wang ZQ, Yang GQ, Zhang DD, Li GX, Qiu JL, Wu J(2024).Isochorismate synthase is required for phylloquinone,but not salicylic acid biosynthesis in rice.aBIOTECH, :1-9. [31]White RF(1979).Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco.Virology, 99:410-412. [32]Wildermuth MC, Dewdney J, Wu G, Ausubel FM(2001).Isochorismate synthase is required to synthesize salicylic acid for plant defence.Nature, 414:562-565. [33]Zhang Y, Li X(2019).Salicylic acid: biosynthesis,perception,and contributions to plant immunity.Curr Opin Plant Biol, 50:29-36. [34]Zhang Y, Yu Q, Gao S, Yu N, Zhao L, Wang J, Zhao J, Huang P, Yao L, Wang M, Zhang K(2022).Disruption of the primary salicylic acid hydroxylases in rice enhances broad-spectrum resistance against pathogens.Plant Cell Environ, 45:2211-2225. [35]Zhao J, Yu N, Ju M, Fan B, Zhang Y, Zhu E, Zhang M, Zhang K(2019).ABC transporter OsABCG18 controls the shootward transport of cytokinins and grain yield in rice.J Exp Bot, 70:6277-6291.