IAA-amido synthetase activity and GH3 expression during development of pea seedlings

Indole-3-acetic acid (IAA) conjugation is an integral component of a sensitive mechanism for the regulation of free auxin level in the cell, in addition to hormone synthesis, degradation and transport. One mechanism of regulating both the level and biological activity of hormone involves the conjugation of IAA to amino acids through IAA-amido synthetases. These enzymes belong to the family of GH3 proteins encoded by early-auxin responsive genes, which are conserved in monocots and dicots. In this investigation, we identified pea IAA-Asp synthetase as a member of the GH3 family proteins based on amino acid sequence determined by tandem mass spectrometry (LC-MS/MS). IAA-Asp synthetase was highly homologous (54-84 % amino acid identity) to the GH3 proteins from Populus trichocarpa, Glycine max, Riccinus communis, Nicotiana tabacum, and Arabidopsis thaliana. Moreover, we studied the subcellular distribution and activity of IAA amido synthetase in developing pea seedlings. The cytoplasmic localization of GH3 IAA amido synthetase was indicated in 12-day-old pea seedlings by immunofluorescence method using anti-AtGH3.5 antibodies. This finding was confirmed by the measurement of the enzyme activity in the subcellular fractions collected by differential centrifugation. The most of IAA-Asp synthetase activity (88 % of the total activity) was detected in the 100,000xg supernatant fluid fraction containing soluble cytoplasmic proteins. 3- and 6-day-old pea seedlings showed a weak IAA-Asp synthetase activity and GH3 mRNA level. Twelve-day-old pea stems exhibited 19-fold, and 4-fold higher levels of transcript and the enzyme activity, respectively than 3-day-old plants. We hypothesized that IAA-amido synthetase expression and activity depend on developmental stage of vegetative tissues and this is associated with changes in free IAA level.