Natural Occurrence of Indoleacetamide and Amidohydrolase Activity in Etiolated Aseptically-Grown Squash Seedlings

Etiolated seedling tissues of aseptically grown squash (Cucurbitamaxima Duch) contain indole-3-acetamide (IAM) as a natural endogenouscompound, conclusively identified by gas chromatography-massspectrometry (GC-MS). Roots of aseptically raised seedlingsalso contain amide hydrolysing activity, which converts IAMto IAA, indoleacetonitrile (IAN) to IAM and IAA, and 1-naphthaleneacetamideto 1-naphthaleneacetic acid. This activity was enriched 48-foldby fractional precipitation with ammonium sulphate, Sephadexgel nitration and anion exchange chromatography. Being hydrolytic,it works equally well in air and in vacuo, without added cofactors.The partially purified enzyme works optimally between pH 7 and7.5, and a K_m value of 80 µM was calculated with IAM asthe substrate. The product of this reaction was definitivelyidentified as IAA by GC-MS. The temperature optimum of thisamidohydrolase lies around 45°C, and it is stable to freezing.A comparison of its properties with the amidohydrolase of Agrobacteriumor crown gall tissue, shows it to be different. In view of thenatural occurrence of both IAM and the amidohydrolase, it issuggested that the IAM pathway of IAA biogenesis is feasiblein etiolated squash seedlings. ⁴Deceased 2/2-1993.