CONJUGATION OF INDOLE-3-ACETIC-ACID (IAA) IN WILD-TYPE AND IAA-OVERPRODUCING TRANSGENIC TOBACCO PLANTS, AND IDENTIFICATION OF THE MAIN CONJUGATES BY FRIT-FAST ATOM BOMBARDMENT LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

Transgenic plants overproducing indole-3-acetic acid (IAA) from expression of the Agrobaterium tumefaciens T-DNA IAA biosynthesis genes were used to study the conjugation of IAA. At the 11-node stage, free IAA, as well as ester- and amide-conjugated IAA, was analyzed in wild-type tobacco SRI and in transgenic plants denoted 35S-iaaM/iaaH (line C) and 35S-iaaM x 35S-iaaH (line X). The transgenic plants contained increased levels of both free and conjugated IAA, and the main increase in IAA conjugates occurred in amide conjugates. Two amide conjugates were identified by frit-fast atom bombardment liquid chromatography-mass spectrometry as indole-3-acetylaspartic acid (IAAsp) and indole-3-acetylglutamic acid (IAGlu), and one ester conjugate was identified as indole-3-acetylglucose. IAAsp and IAGlu were also identified as endogenous substances in wild-type plants. In wild-type plants, the percent of total IAA in the free form was significantly higher in young leaves (73 +/- 7%, sD) than in old leaves (36 +/- 8%), whereas there was no difference between young (73 +/- 8%) and old internodes (70 +/- 9%). In IAA-overproducing transformants, both free and conjugated IAA levels were increased, but the percent free IAA was maintained constant (57 +/- 10%) for both leaves and internodes, independent of the total IAA level of tissue age. These results suggest that synthesis or transport of IAA conjugates is regulated in the vegetative wild-type plant, and that different organs possess a unique balance between free and conjugated IAA. The IAA-overproducing plant, however, acquires a lower proportion of free IAA in the stem and younger leaves, presumably determined by a higher conjugation in those tissues compared with wild type.