Criteria for Assessing the Accuracy of the Pyrone Fluorimetric Assay for Indole-3-acetic Acid

Abstract

The effects of scattered light and the presence of contaminating fluorophores on the sensitivity and accuracy of the pyrone fluorimetric assay for indole-3-acetic acid (IAA) were investigated. With extracts from plant tissues sources of ambiguity in the fluorescence measurements were established. Criteria, based on the fluorescence spectra and kinetics of change of fluorescence intensity of the pyrone derivative, are presented for assessing the accuracy of IAA estimations using this technique. INTRODUCTION The reaction of indole-3-acetic acid (IAA) with acetic anhydride to form the fluorescent derivative 2-methylindolo-2,3:3',4'-pyr-6-one (2-MIP) provides, in principle, a straightforward estimation of nanogram levels of IAA in partially purified plant extracts (Stoessl and Venis, 1970; Knegt and Bruinsma, 1973). Recent investigations, however, have shown that the reaction is not specific for IAA, but rather exhibits a selectivity for a range of acidic and neutral indole compounds that possess a C3-methylene side-chain (Boettger, Engvild, and Kaiser, 1978; Mousdale, Butcher, and Powell, 1978). In addition, measurements can be seriously distorted by light-scattering effects caused by uncharacterized compo nents present in IAA-containing fractions purified from plant tissue extracts (Eliasson, Stromquist, and Tillberg, 1976; Kamisaka and Larsen, 1977; Mousdale et al., 1978). In view of these findings, the investigation of the accuracy of the pyrone fluorimetric assay was deemed necessary, and resulting detailed criteria for assessing the accuracy of assay measurements are described. MATERIALS AND METHODS Fruit and vegetative tissue samples from apple (Malus domestica Borkh. rootstock M.9 and cv. 'James Grieve' on rootstock M. 26), plum (Prunus domestica L. cv. 'Victoria'), and broad-leaved lime (Tilia platyphyllos Scop.) were collected from both conventional orchard and hedgerow-grown trees at East Mailing Research Station. Freeze-dried material of apple (cv. 'Miller's Seedling') leaf and tobacco (Nicotiana tabacum L. cv. 'Xanthi-nc') stem were provided by Dr. R. M. Fulford (Physiology Department, East Mailing) and Dr. A. Cassells (Department of Horticulture, Wye College, University of London), respectively. Methods for the extraction and purification of IAA from plant tissues and for the fluorescence measurements were as previously described (Mousdale et This content downloaded from 207.46.13.111 on Tue, 09 Aug 2016 06:29:58 UTC All use subject to http://about.jstor.org/terms 516 Mous dale—Fluorimetric 1AA Assay ai, 1978). Fluore scene; spectra were recorded with a Baird-Atomic (Braintree, U.K.) 'Fluoripoint' spectrofluorimeter fitted with a high-intensity Xenon light source; excitation and emission monochromator band widths were 10 nm. Absorption measurements were made with a Pye Unicam (Cambridge, U.K.) model SP8-100 spectrophotometer. IAA (free-acid form) was purchased from Sigma, London, Chemicals Ltd., Poole, U.K.; acetic anhydride (Analar grade) from Fisons Ltd., Loughborough, U.K.; and trifluoroacetic acid (Special for Spectroscopy grade) from British Drug Houses Ltd., Poole, U.K. RESULTS Precision of fluorescence measurements The effects of sampling and other random errors on the accuracy of the assay were tested using nanogram and sub-nanogram quantities of IAA. The major factor limiting accuracy was found to be a transient light-scattering which occurred when reaction mixtures containing trifluoroacetic acid as catalyst were diluted with distilled water to terminate the reaction (Fig. 1). This effect was caused by the formation of fine bubbles in the solution, and could be eliminated by delaying the start of the emission spectrum scanning procedure until 1-5 min after adding water to the reaction mixture. With this precaution, sub-nanogram quantities of IAA could be detected with good reproducibility and with a linear relation between fluorescence intens ity and quantity of IAA reacted (Table 1). Contributions from other fluorophores 2-MIP has characteristic fluorescence excitation and emission spectra (Mousdale et al., 1978). Identical emission spectra and similar excitation spectra were recorded when applying the assay to IAA-containing fractions from plant tissue extracts (Table 2). However, with the exception of the tobacco tissue