The biosynthesis of indoleacetic acid and its regulation

Abstract

Growth studies on Phaseolus vulgaris L., cv Tendergreen were carried out and it was found that the growth curve of the seeds was triple sigmoid in shape. The bean pods with different seed numbers had a normal distribution and most pods retained had five to six seeds. The increase in the bean pod length was completed at an early stage. The growth rate of fresh weight of the pod wall was also fast at the beginning of the bean development. When this growth rate decreased, the growth rate in the seed fresh weight increased. Changes of protein concentrations in the bean seeds had the same pattern as the seed growth, but slightly preceded it, and had a main peak at the early stage of the seed development. Using gas chromatography-mass spectrometer (GC-MS) method indole-3-acetic acid (IAA) and indole-3-ethanol (IEt) were identified as native components in the bean seeds. Changes in IAA and IEt content during development of bean seeds of Tendergreen were determined using high-resolution selected ion monitoring (SIM) GC-MS. The highest concentration of IAA in the seeds was found at the early stage of the bean growth, which was associated with the rapid increase in weight of the pod wall, and then with the fast increase in seed weight. When the IAA concentration in the seeds decreased to its steady low level, the seed growth rate started to decrease. There was closer correlation of the changes in seed IAA concentration with the changes in seed protein concentration than with the changes in seed fresh weight. The changes in the seed IEt concentrations had the same trends as those of the seed IAA concentrations, but slightly preceded the changes in IAA. The extent of the changes in the IEt level varied under different growing conditions, while the extent of the changes of the IAA level remain almost the same. The endogenous pool of IAA was much larger than that of IEt in the bean seeds. The IEt oxidative capacity of an ammonium sulfate precipitate of the Tendergreen bean seed extracts was also greatest at the early stage of the seed development, and the changes in this capacity had a similar pattern to changes in seed IEt and IAA levels, coincided with the changes in IEt level, but occurred slightly earlier than the changes in IAA levels. Purification of IEt oxidase was carried out from two bean cultivars, Farden Losa and Labrador. IEt oxidase from the two cultivars showed some different properties, such as molecular weights and pI values. The IEt oxidase was purified more than 1000-fold to apparent homogeneity from the Labrador bean cultivar. This enzyme had a molecular weight of 56 kD and a pH optimum 6-7. The enzyme reaction required oxygen, but was not stimulated by either NADP (0.2 mM) or FAD (1 mM). Production of hydrogen peroxide was observed with the reaction. 10 mM EDTA and iodoacetate inhibited the enzyme reaction. The enzyme was strongly inhibited by 0.25 mM gibberellic acid (GA3), and stimulated by phenol at a concentration of 10 mM. At a concentration of 0.2 mM, inhibition by indole-3-acetaldehyde (IAAld) was observed, while inhibition by IAA was not obvious. A rabbit antibody was raised against the IEt oxidase from Farden Losa bean seeds after resolution on SDS gel electrophoresis. Using the technique of Immunoblotting (Western blot) and 1251-protein A binding, detection of changes in the amount of the antibody-binding protein during the seed development was attempted. Non-specific binding was encountered which was probably due to the interference of lectins present in the seeds. This was confirmed by immunodiffusion experiments. Some possibilities for the regulation of IAA biosynthesis are discussed.