Hydrolysis of Indole-3-Acetic Acid Esters Exposed to Mild Alkaline Conditions

Abstract

Ester conjugates of indole-3-acetic 16,18,22,23) following various protocols suggested that a more systematic evaluation of this process was needed. In this report we present data on the kinetics of hydrolysis of reagent IAA-GLU2 at selected pH values and compare the lability of IAA-GLU to that of IAA-MI and several other ester conjugates of IAA. In addition, we report the behavior of a relatively polar conjugate, IAA-GLU, and a relatively apolar conjugate, IAA-PNP, in the 'three phase' system described by Liu and Tillberg (16). These data indicate that problems with inadvertent hydrolysis are apparent even at moderate pH values and in multiphase extraction systems.IAA is present in plant tissues as the free acid and also in the form of covalently linked conjugates (5,7,14,20). Two classes of conjugates, based on their alkaline lability, have been recognized. Ester conjugates (including ester, acyl anhydride, and S-acyl anhydrides) are hydrolyzed easily in basic solutions. Initial studies of auxin extraction techniques (1-3, 12-15, 21) established that IAA conjugates could be hydrolyzed during extraction, either by apparently enzymatic mechanisms or by exposure to alkaline environments. For example, Berger and Avery (2, 3) used treatment at pH 9.6 for 5 min to liberate free IAA from its 'complex' in maize kernels. At the time many of these classic studies were done the identity of the bound forms of IAA were unknown. However, since that time many endogenous auxin conjugates have been isolated and structurally characterized (7,20).The presence ofthese bound forms and their lability dictates that in analysis of the free hormone content of plant tissues

Quantification of Free Plus Conjugated Indoleacetic Acid in Arabidopsis Requires Correction for the Nonenzymatic Conversion of Indolic Nitriles

“…Severa1 workers have determined the conditions necessary for quantitative hydrolysis of such conjugates (Bandurski and Schulze, 1977;Cohen and Bandurski, 1982;Cohen et al, 1986). Thus, treatment with 1 N NaOH for 1 h at room temperature will result in the quantitative hydrolysis of ester forms with no measurable hydrolysis of amide conjugates (Baldi et al, 1989). Amide forms require more rigorous hydrolysis and are cleaved quantitatively by 7 N NaOH for 3 h at 100°C (Bialek and Cohen, 1989).…”

Low-Fluence Red Light Increases the Transport and Biosynthesis of Auxin

“…For the rest of the homogenate, 10 mL was hydrolyzed in 1 N NaOH (1 h, room temperature; Baldi et al, 1989) for measurement of free plus ester-linked IAA and 10 mL was hydrolyzed in 7 N NaOH (3 h, 100°C under N 2 ; Bialek and Cohen, 1989) for measurement of total IAA (free plus ester-linked and amidelinked IAA). After hydrolysis, the pH of the hydrolysate was adjusted to 2.7, and the free IAA plus that released from the conjugates was purified from the extracts using TopTips containing 7.5 mg of C 18 resin (12213020; Varian), washed twice with 60 mL of water, eluted twice with 50 mL of methanol, collected in 250-mL glass inserts, and methylated as described above.…”

Transport of Indole-3-Butyric Acid and Indole-3-Acetic Acid in Arabidopsis Hypocotyls Using Stable Isotope Labeling

“…The major fragment ion for both 3-substituted indoles involves a ring expansion to form the relatively stable quinolinium ion in high yield (Marx and Djerassi, 1968 . As shown in Figure 5H, in the lower section and receiver, the level of Free IAA and IBA can be released from their conjugated forms via chemical hydrolysis: weak base hydrolysis breaks the ester linkage (Baldi et al, 1989), while strong base hydrolysis breaks both ester and amide linkages (Bialek and Cohen, 1989 (Fig. 6B).…”