Improved Procedure for the Estimation of Nanogram Quantities of Indole-3-acetic Acid in Plant Extracts using the Indolo-α-pyrone Fluorescence Method

Abstract

The indolo-a-pyrone fluorescence method of determining indole-3-acetic acid (IAA) is improved by adding butylated hydroxytoluene (BHT), an antioxidant, to samples: addition of BHT increases the fluorescence intensities and decreases their variability so that amounts of IAA as small as 0.1 to 1 nanogram become measurable. Interfering compounds, 4-chloroin-dole-3-acetic acid and 5-hydroxyindole-3-acetic acid, can be separated from [AA by thin-layer chromatography using polyamide as the solid support, and benzene-ethyl acetate-acetic acid (70:25:5, v/v) as the developing solvent. Polyamide thin-layer chromatography is also superior in purifying IAA without significant loss or decomposition. A serious problem in extracting IAA from plant tissues is the considerable loss. Losses can result from decomposition of IAA when its solutions in organic solvents are evaporated to dryness, apparently due to some impurity in the solvents. Decomposition can be eliminated by simple treatments of the solvents, Le. addition of BHT, washing with water, and passage through cotton wool. The latter is particularly effective in the case of ethyl acetate and ether. Loss of IAA by photodecomposition is also substantial even in dried samples. Ways to minimize the conversion of indole-3-pyruvic acid to IAA during extraction procedure are also described. A revised procedure of IAA extraction and determination is proposed. Applied to etiolated shoots of maize seedlings, it enables content to be estimated (18 nanograms per gram fresh weight) with over 90% recovery in terms of I14CIIAA added at the beginning of the extraction. Several physicochemical methods for determining IAA in the ng range have been proposed in the last decade to replace bioassay systems but, for various reasons, they are of limited use for plant extracts and are not readily applicable to physiological experiments (24). The indolo-a-pyrone fluorescence method, which was first devised by Stoessl and Venis (23), has been shown to be highly sensitive and specific (17, 23) and used to determine IAA in crude acidic extracts prepared by liquid partitioning (6, 10, 11, 23). This procedure suffers from the disadvantage that 4-Cl-IAA' and 5-OH-IAA, which occur naturally in plant tissues (8, 15, 25) can produce fluorescence indistinguishable from that of IAA after 'Abbreviations: 4-C1-IAA, 4-chloroindole-3-acetic acid; 5-OH-IAA, 5-hydroxyindole-3-acetic acid; IPyA; indole-3-pyruvic acid; TFAA, trifluo-roacetic acid; BHT, butylated hydroxytoluene (2,6-di-tert-butyl4-methyl-phenol). conversion to indolo-a-pyrone (4). The procedure also suffers from the shortcoming that it cannot readily be applied to certain plant tissues, e.g. etiolated tissues of maize or wheat, because of severe interference by impurities present in the extracts (17). If adequate purification steps are included to remove 4-Cl-IAA, 5-OH-IAA, and other interfering substances, the fluorescence method may provide a routine, simple procedure of estimating ng amounts of IAA in plant tissues. This paper describes such a procedure. The considerable loss of IAA during its extraction has long been a problem (5, 9, 12, 13, 22). To estimate IAA in plant tissues using the fluorescence method, [14C]IAA can be used as an internal standard to correct for losses (11). Inasmuch as the losses were attributed to decomposition of IAA, there is a possibility that some breakdown derivatives of labeled IAA contribute to the recovered radioactivity, leading to erroneous estimation of recovery. Here, causes of IAA decomposition are indicated and ways to overcome them are suggested. A potential source of error in the accurate estimation of IAA in plant extracts can also arise from the conversion of IPyA to IAA. Atsumi et al. (1, 2) considered this to be a serious problem with senescent tissues. Its magnitude is unknown for growing tissues because of ignorance of their content of IPyA. Nevertheless, the biosynthetic pathway from tryptophan to IAA via IPyA has been established (21). IPyA conversion has to be taken into consideration in assessing any extraction procedure not known to prevent effectively or to avoid the conversion of IPyA to IAA. We have investigated the occurrence of this conversion during the extraction procedures and suggest ways to minimize it. We describe improved procedures of IAA extraction and determination which, when applied to etiolated shoots of maize, allowed estimation of IAA with a total recovery of more than 90%.