Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea
灰葡萄孢菌感染拟南芥叶片后Camalexin的定量测定
发布: 2015年01月20日第5卷第2期 DOI: 10.21769/BioProtoc.1379 浏览次数: 11321
评审: Tie LiuSollapura J. Vishwanath
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参见作者原研究论文
The authors used this protocol in:
Jul 2014
Abstract
Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007).
The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography–mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.
Figure 1. The structural formula of camalexin
Materials and Reagents
- Arabidopsis leaves
- Liquid nitrogen
- Dichloromethane (DCM) (Sigma-Aldrich, catalog number: 270997 )
- Methanol (LC/MS grade) (Carlo Erba Reagents, catalog number: 414831 )
- Formic acid (gradient grade) (Sigma-Aldrich, catalog number: F0507 )
- Water with 0.1% formic acid (LC/MS grade) (Sigma-Aldrich, catalog number: 34673 )
- Camalexin standard (Sigma-Aldrich, catalog number: SML1016 )
- Extraction buffer (see Recipes)
Equipment
- Mortar and pestle
- Spatula
- Glass Pasteur pipettes
- Tube adapters
- Screw cap round bottom borosilicate glass culture tubes (16 x 125 mm) (Pyrex) (Sigma-Aldrich, catalog number: Z653594-40EA )
- Disposable borosilicate glass culture tubes (Pyrex)
- Spin-X centrifuge tube filters with nylon membranes (Sigma-Aldrich, catalog number: CLS8170-200EA )
- Glass vials with cap (capacity: 100 µl)
- Vortex mixer
- Rocking shaker
- Nitrogen evaporator (e.g. sample concentrator with dry block DB-3D) (Techne) (Figure 1)
- Balance
- Variable speed refrigerated centrifuge with rotor having number of rotor cavities x nominal largest load (capacity: 8 x 50 ml) (e.g. Beckman Coulter)
- HPLC (e.g. Thermo Fisher Scientific, model: UltiMate 3000 HPLC )
- High performance reversed-phase columns C18 for the separation of small molecules (e.g. Acclaim120 C18 Reverse-phase column-3 μm, 200 Å, 2.1 x 150 mm) (Thermo Fisher Scientific, catalog number: 059130 )
- ESI-Mass Spectrometer (e.g. Orbitrap XL Discovery, Thermo Fisher Scientific)
Software
- Analysis software (e.g. Thermo Xcalibur software 2.10, Thermo Fisher Scientific)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Savatin, D. V., Bisceglia, N. G., Gravino, M., Fabbri, C., Pontiggia, D. and Mattei, B. (2015). Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea. Bio-protocol 5(2): e1379. DOI: 10.21769/BioProtoc.1379.
- Savatin, D. V., Bisceglia, N. G., Marti, L., Fabbri, C., Cervone, F. and De Lorenzo, G. (2014). The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-Related Protein Kinases Are Required for Elicitor-Induced Oxidative Burst and Immunity. Plant Physiol 165(3): 1188-1202.
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分类
植物科学 > 植物免疫 > 信号感知与传递
植物科学 > 植物生物化学 > 其它化合物
微生物学 > 微生物-宿主相互作用 > 体内实验模型
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