A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-
- PDF / 416,685 Bytes
- 9 Pages / 547.087 x 737.008 pts Page_size
- 11 Downloads / 180 Views
SHORT COMMUNICATION
A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-3-acetic acid Michał Szkop • Wiesław Bielawski
Received: 26 August 2012 / Accepted: 18 October 2012 / Published online: 31 October 2012 Ó The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (kex = 280/kem = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625–125 lg mL-1 (r2 C 0.998) and the limits of detection were below 0.015 lg mL-1. The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants. Keywords HPLC Indolic compounds IAA biosynthesis
M. Szkop (&) W. Bielawski Department of Biochemistry, Faculty of Agriculture and Biology, Warsaw University of Life Sciences– SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland e-mail: [email protected]
Introduction Indole-3-acetic acid (IAA) is the most abundant naturally occurring auxin, which is a class of phytohormones implicated in the regulation of plant growth and development. In addition to plants, diverse species of soil and plant-associated bacteria can also produce IAA. Through the identification of bacterial genes and enzyme activities and the detection of indolic intermediates in bacterial culture supernatants, the existence of five different IAA biosynthesis routes has been determined with tryptophan (Trp) as a precursor: the indole-3-pyruvate (IPyA), indole-3-acetamide (IAM), tryptamine (TAM), indole-3-acetonitrile (IAN) and the Trp side chain oxidase pathways (Fig. 1; described in Carreno-Lopez et al. 2000; Spaepen et al. 2007). Additionally, it was observed that IAA produced by bacteria via distinct pathways may differentially affect plant growth. While IAA biosynthesized by beneficial bacteria designated as PGPR via the IPyA intermediate stimulated root proliferation and growth, IAA produced by phytopathogenic bacteria via the IAM intermediate induced galls and tumors formation in plants (Lambrecht et al. 2000). These findings suggest that there is a link between the IAA biosynthetic pathway used by a given bacterial strain and the host plant phenotype. It is therefore important not only to verify the ability of a particular strain of bacteria to produce IAA but also to investigate the biosynthetic route of this compound through, together with above mentioned approaches, determination of other
123
684
Antonie van Leeuwe
Data Loading...
