The in vitro biosynthesis of indole-3-butyric acid (IBA) was investiga
ted in maize (Zea mays L.). Incubation of microsomal membranes from sh
oots and roots of dark-grown maize with labelled indole-3-acetic acid
(IAA), acetyl coenzyme A (acetyl CoA) and ATP resulted in the formatio
n of an unknown labelled reaction product, product X. Other coenzymes
had very little effect on the formation of this product. No IBA was de
tected in this system. The labelled product was purified and fed to ot
her cell fractions of dark-grown maize. Incubation with the organelle
fraction using NADPH as a cofactor resulted in the conversion of produ
ct X to a new compound which had the retention time of IBA in HPLC. At
tempts to identify product X led to the hypothesis that it is a conjug
ate of IAA with ADP. Microsomal membranes from light-grown shoots and
roots converted IAA to IBA, as demonstrated by HPLC analysis. The iden
tity of the IBA peak was confirmed by GC-mass spectrometry. Microsomal
membranes from light-grown roots formed both IBA and little of produc
t X. For the direct in vitro biosynthesis of IBA acetyl CoA was needed
as a cofactor, and the reaction as enhanced by the addition of ATP or
NADPH. The optimum pH of the in vitro formation of IBA was 6-7 in the
roots and 5 in the shoots. Product X was formed only at neutral pH (7
-8). The K-m value for IAA in the synthesis of IBA was 16 mu M.