Abstract
Indole-3-acetic acid (IAA) a phytohormon of auxin type is synthesized by different ways in plants and some bacteria (Agrobacteria, Pseudomonas and some others). The enzyme indolylacetamide hydrolase transforms indolylacetamide to IAA mainly in bacteria. However, recently published data showed that some plants can also hydrolyze indolylacetamide into IAA. In order to elucidate the role of indolylacetamide as an auxin precursor in plants and bacteria, productive method of determination of the activity of indoleacetamide hydrolase is necessary. The simple, inexpensive and productive method for the measurement of indoleacetamide hydrolase activity was elaborated based on significant difference between IAAM and IAA in color developed with Salkovski’ reagent. The light absorbance increased during conversion of IAAM to IAA by protein extracts from some plant cells and this increase may be used for quantitative estimation of indoleacetamide hydrolase activity. The method is suitable for fast discovery of indoleacetamide hydrolase activity before planning more complicated analyses and for the analysis of many probes at a short time in physiological and biochemical experiments. A detailed protocol for determination of indoleacetamide hydrolase activity by the elaborated method is described.
Highlights
Indole-3-acetic acid (IAA) is a multifunctional phytohormone playing important role in the regulation of plant growth and development
Colors developed after mixing IAA and IAAM solutions with Salkowsky’s reagent had an absorption maximum at 530 nm, but it was 3.4 times higher for IAA than for IAAM. It means that amount of absorption will be increased during conversion of IAAM to IAA in the course of IAAMH action because molar coefficient of absorption at 530 nm (α530) was 1.63 × 10−3 × mM−1 for IAAM and 5.52 × 10−3 × mM−1 for IAA
The formula is proposed for quantitative estimation of IAAMH activity:
Summary
Indole-3-acetic acid (IAA) is a multifunctional phytohormone playing important role in the regulation of plant growth and development. These bacteria have two genes: one for tryptophan-2-monooxigenas (aux1) and other one for IAAMH (aux). There are many other amidohydrolases which can hydrolyse IAA amino acid conjugates [10] and other amides Genes coding these amidohydrolases may have aux2-like but not identical sequences. There may be two final proofs of IAAMH activity in plants: the ability of IAAM to be a substitute of IAA and other auxins in plant growth regulation and the ability of plant extracts to transform IAAM to IAA
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