Changes in the Level of Free and Ester Indol-3yl-Acetic Acid in Growing Maize Roots

Abstract

ABSTRACEThe levels of free and ester-linked indole-3-acetic acid (IAA) in different parts of the maize root were measured using gas chromatographymass spectrometry (selected ion monitoring). In roots of 2-day-old plants, the distribution of free and ester IAA differed both along the root and between stele and cortex. The levels of IAA and IAA esters were then measured in whole roots and in the elongation zone using roots ofdifferent ages. The level of ester IAA decreased steadily with time. In contrast, the level of free IAA in the elongation zone was found to increase after a few days of culture at which time the rate of root growth was decreasing.The role of IAA in the regulation of the growth and development of roots has been extensively reviewed (2,24).Several reports have described the identification and quantification of IAA in maize roots using combined GC-MS (7, 21). These studies have shown that most of the IAA was localized in the stele (7,9,23) and that the root cap also contained large amounts of IAA (21).The amount of IAA which was liberated from esters by alkaline hydrolysis has also been determined in maize seedlings (5, 1 1) and recently in maize roots (14). Several roles for esters of IAA have been proposed. They could be a source of IAA for seed and seedling during germination (10) or be concerned in hormonal homeostasis in the shoot (6). Moreover, esterification could play a protective role in preventing peroxidative attack (8).It has recently been shown that when maize roots were sorted into classes according to their growth rate, there was an inverse correlation between the growth rate and the level of IAA in the elongation zone (18). Thus, the aim of the present investigation was, using GC-SIM'-MS, to determine the level of free and esterified IAA, during germination and growth, in whole roots and in the elongation zone. MATERUILS AND METHODSMaize Seedlings. The germination of selected caryopses of Zea mays cv LG 11 (Association Suisse des Selectionneurs, Lausanne) has been previously described (16 first set of experiments, 2-d-old intact seedlings with straight primary roots (16 ± 1 mm in length) were selected and cut into four sections: the cap (0-0.5 mm), the meristem (0.5-1.0 mm), the elongation (1.0-5.0 mm), and the differentiation zone (5-15 mm). For some of the roots, the stele and the cortex were separated after removing the apical 5 mm and cut giving a segment of 9 mm in length.Plant Material for Time-Course Analyses. In these assays, whole roots were harvested after different periods of culture. First, they were collected for length, fresh weight, and (after freezedrying) dry weight measurements. Selected roots (as described above) were harvested for the determination of endogenous IAA in the whole root or in the elongation zone (2.5-5.0 mm; 18, 25).Growth Classes. Two-d-old intact maize seedlings (15 ± 1 mm in root length) were placed vertically for 8 ± 1 h in closed boxes (90 ± 5% RH). Photographs were taken initially and 8 h later (18). Growth analyses were performed using phot...

Quantification of indole-3-acetic acid in untransformed and Agrobacterium rhizogenes-transformed pea roots using gas chromatography mass spectrometry

“…The extraction of free IAA was done according to Saugy and Pilet (1987) and Meuwly and Pilet (1991), with some minor modifications. To the dried plant material were added 20 ng [2Hs]IAA as internal standard and 3 ml of ice-cold sodium acetate-acetic acid buffer (10 mM, pH 4.8).…”

Simultaneous Gas Chromatography-Mass Spectrometry Quantification of Endogenous [12C]- and Applied [13C]Indole-3yl-Acetic Acid Levels in Growing Maize Roots

“…Extraction was essentially as previously described (25,26,29) except a prepurification step was employed using Sep-Pak (26), and 6 ml of ice-cold sodium acetateacetic acid buffer (I1/15 M, pH 4.0) were added to dried plant material and the sample was homogenized (vortex, 15 s) and partitioned against 2.5 mL of ethylacetate. The partition (15 min, rotative stirring) was repeated twice and the organic layer was evaporated (40°C) to dryness under a stream of N2.…”

“…In this current work, a stable isotope of IAA (labeled with 6 atoms of 13C) was therefore employed not as the more usual internal standard (7) but for hormone treatments of growing maize roots. The aim of the present investigation was to determine simultaneously, using GC-SIM2-MS, the levels of endogenous ['2C]IAA and applied ['3C]IAA in three different zones (apical, elongating, differentiating) and thus to correlate the amount of the two forms of IAA to root growth.Several reports have established, using GC-MS, the natural occurrence of IAA and its role in the regulation of root growth (5,8,22,25,29). Two main approaches to this problem have generally been used.…”

“…Both effects depend on the amount of IAA (22) and the localization of the treatment along the root (14). Second, quantifications of levels of endogenous IAA in maize roots have shown an inverse relation (modified during germination) between the growth rate and the level of free and ester-bound IAA in the elongation zone (22,29). …”

“…Several reports have established, using GC-MS, the natural occurrence of IAA and its role in the regulation of root growth (5,8,22,25,29). Two main approaches to this problem have generally been used.…”