Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea

• Published in: Bio-protocol Vol. 5; no. 2
• Main Authors: Savatin, Daniel V, Bisceglia, Nora Gigli, Gravino, Matteo, Fabbri, Claudia, Pontiggia, Daniela, Mattei, Benedetta
• Format: Journal Article
• Language: English
• Published: United States Bio-protocol LLC 20.01.2015
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.1379

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 , from which it takes its name, infected with (Browne , 1991). Production of camalexin is also induced in leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja , 2012) as well as by abiotic stresses, such as UV and chemicals ( acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao , 1998; Tierens , 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 , 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.