pubs.acs.org/JAFC Published on Web 11/03/2009 © 2009 American Chemical Society 10518 J. Agric. Food Chem. 2009, 57, 10518–10523 DOI:10.1021/jf9024008 Simple and Rapid Capillary Zone Electrophoresis Method for the Detection of Coronamic Acid, a Precursor to the Pseudomonas syringae Phytotoxin Coronatine ASWATHY SREEDHARAN, † ALEJANDRO PENALOZA-VAZQUEZ, † MA.CRISTINA ESCOBER, ‡ CAROL L. BENDER,* ,† AND PATRICIA RAYAS-DUARTE* ,‡,§ † Department of Entomology and Plant Pathology and ‡ Robert M. Kerr Food & Agricultural Products Center and § Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078 The phytotoxin coronatine (COR) is produced by various pathovars of the plant pathogen Pseudomonas syringae, which infects a wide variety of crops. COR consists of two distinct moieties, coronafacic acid (CFA) and coronamic acid (CMA), which are derived from a modified polyketide pathway and isoleucine, respectively. Mutants defective in the CMA or CFA structural gene clusters have been used to study COR biosynthesis, and these mutants are commonly characterized using high-performance liquid chromatography (HPLC). Although the same extraction and HPLC method can be used for detection and quantification of COR and CFA, the detection of CMA by HPLC requires different fractionation and HPLC separation procedures, which are tedious and labor intensive. In this study, we used capillary zone electrophoresis (CZE) as a fast and accurate detection method for the quantification of CMA present in the culture supernatant of P. syringae pv. glycinea (Psg) PG4180 and P. syringae pv. tomato (Pst) DC3000. Analysis was performed by CZE using 100 mM phosphate buffer (pH 2.5) as a separating buffer, an applied voltage of 12 kV, and UV detection at 214 nm. Selected mutants defective in COR biosynthesis were used to validate CZE as a detection method. CMA production by Psg strain 18a/90, which lacks the COR gene cluster, and derivatives of 18a/90 was also evaluated. Furthermore, a procedure for the extraction and detection of CMA present inside the cells of Psg 18a/90 was developed. In conclusion, CZE was shown to be a rapid and sensitive method for the detection and quantification of CMA in P. syringae. KEYWORDS: Capillary zone electrophoresis; coronatine; cornonamic acid; Pseudomonas syringae INTRODUCTION Coronatine (COR) is a nonhost specific phytotoxin produced by various pathovars of Pseudomonas syringae including atro- purpurea, glycinea, maculicola, morsprunorum, and tomato, which infect ryegrass, soybean, crucifers, Prunus spp., and tomato, respectively. Depending on the plant host, this toxin is known to elicit chlorosis, inducing hypertrophy, inhibit root elongation, and stimulate ethylene production ( 1 , 2 ). COR shares structural and functional similarities with jasmonic acid, an endogenous signaling molecule in plants that functions as a growth regulator ( 3 -5 ). COR impacts the outcome of the plant stress response associated with pathogens and herbivory and also modulates plant responses to environmental stresses ( 6 ). In recent years, COR has attracted considerable interest because of its role in the plant defense response and its potential use in various commercial applications. COR has been shown to alleviate salinity stress in cotton ( 7 ) and has potential use as an abscission agent in the harvest of mature citrus fruits ( 8 ). COR consists of two distinct moieties, coronamic acid (CMA) and coronafacic acid (CFA) (Figure 1A)( 9 ), which are derived from two entirely different biosynthetic pathways. CFA is bio- synthesized via a modified polyketide pathway ( 10 ), whereas CMA is an ethylcyclopropyl amino acid that originates from isoleucine ( 11 ). CMA is generated from L-allo-isoleucine by a nonribosomal peptide synthetase ( 12 ). CFA and CMA are coupled by an amide bond to form COR ( 11 , 13 ), and the enzyme involved in this reaction lacks rigid specificity for the amino acid substrate. Hence, in addition to COR, various other CFA-amino acid complexes are biosynthesized including cor- onafacoylisoleucine, coronafacoylalloisoleucine, and coronafac- oylvaline ( 14 -17 ). Among the analogues, COR is the most toxic coronafacoyl compound made by COR-producing organ- isms ( 15 ). However, studies have shown that both CFA and CMA are important in the biological activity of COR ( 5 , 18 ). *To whom correspondence should be addressed. (P.R.-D.) 107 FAPC, Department of Biochemistry and Molecular Biology, Robert M. Kerr Food & Agricultural Products Center, Oklahoma State University, Stillwater, OK 74078. Phone: (405) 744-6468. Fax: (405) 744-6313. E-mail: pat.rayas_duarte@okstate.edu. (C.L.B.) 127 Noble Research Center, Department of Entomology & Plant Pathology, Oklahoma State University, Stillwater, OK 74078. Phone (405) 744- 9945. Fax (405) 744-7373. E-mail: carol.bender@okstate.edu.