Quantitation of Indoleacetic Acid Conjugates in Bean Seeds by Direct Tissue Hydrolysis

Abstract

Gas chromatography-selected ion monitoring-mass spectral analysis using [13Cs]indole-3-acetic acid (IAA) as an internal standard provides an effective means for quantitation of IAA liberated during direct strong basic hydrolysis of bean (Phaseolus vulgaris L.) seed powder, provided that extra precautions are undertaken to exclude oxygen from the reaction vial. Direct seed powder hydrolysis revealed that the major portion of amide IAA conjugates in bean seeds are not extractable by aqueous acetone, the solvent used commonly for IAA conjugate extraction from seeds and other plant tissues. Strong basic hydrolysis of plant tissue can be used to provide new information on IAA content.age of acetone greatly improved the yield. Without the ability to determine a true value for 'total IAA' in plant tissues, the presence of such diverse molecular species is difficult to ascertain. In addition to these considerations, in our studies we were also interested in extending our investigations to other plant tissues and to other species (see, for example, refs. 2 and 5), where the solubility of macromolecular conjugates, if present, are totally unknown.In this report we describe the direct use of strong basic hydrolysis on seed powder without prior extraction in order to study the total IAA in bean seeds. Simple techniques are described that help to avoid artifacts due to the conversion of indolic compounds (other than conjugates) to IAA in such crude hydrolysates. MATERIALS AND METHODSQuantitative studies of IAA conjugates can be carried out by two approaches, the direct quantitation of specific conjugates (7, (Fig. 1) purged with water-saturated N2 (99.998%, Air Products). Halfofthe samples were hydrolyzed in the presence of nitrogen that had been repurified using an O2 scrubbing cartridge (Oxisorb, MG Scientific Gases) added to the gas line. After hydrolysis, hydrolysates were diluted with 5 mL water, acidified with HC1 to pH 2.5, and centrifuged at l0,OOOg for 5 min.The purification of hydrolysates prior to GC-MS analysis included Fisher PrepSep C18 minicolumn clean-up and HPLC, according to the procedure developed in our laboratory (6). First, the samples were passed through a conditioned (washed with 5 mL ofhexane, methanol, water, and 1 % acetic

A gene encoding a protein modified by the phytohormone indoleacetic acid

“…In most studies done over the last 50 years, the amount of auxin obtained after solvent extraction did not account for the total IAA found following exhaustive extraction over time (31,32). More recent studies showed a similar phenomenon, that the amount of total IAA (free plus conjugated) obtained by direct hydrolysis of the tissue was much greater than the amount readily extractable with solvents (12,33,34). This difficulty in accounting for the IAA present in plants has only recently been resolved by the isolation of proteins and peptides to which IAA was attached.…”

“…10 and 11). The amount of IAA released by alkaline hydrolysis of the protein was calculated by the isotope dilution equation (10,12). Molecular Mass Determination of IAP1.…”

“…10). The Arabidopsis seed 35-kDa immunostaining protein was partially purified and subjected to alkaline hydrolysis (10)(11)(12). GC-MS analysis confirmed the presence of IAA co- The iap1 cDNA was expressed in E. coli.…”

“…The linkage between auxin activity and protein metabolism seems to be reinforced by a diverse array of experimental approaches, and the presence of specific proteins to which IAA is attached may have significance beyond the important regulatory roles already assigned to conjugates for modulation of IAA levels. Regardless of the broader consequences of protein modification, the description of IAA proteins should allow a more detailed picture of IAA metabolism, especially in plants such as bean and Arabidopsis where total IAA levels greatly exceed the IAA attributed to the free hormone and solvent-extractable conjugates (12,23).…”

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

“…The bean seeds used in our studies contain large amounts of IAA conjugates, especially amide-linked conjugates: about 600 ng . g-1 fresh weight or 150 ng/seed at full seed maturity (8,9). Their levels decline in the first days of germination but remain relatively high through the later period of germination (11).…”