Compartmentation of indole-3-acetic acid metabolism in protoplasts isolated from leaves of wild-type and IAA-overproducing transgenic tobacco plants

Abstract

Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [¹⁵N₁]IAA synthesised from [¹⁵N₁]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SRI plants, and in IAA-overproducing plants expressing the Agrobacterium tumefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was located exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conversion into IAA by action of the iaaH-encoded hydrolase. The free-IAA level, however, was raised only 8% in iaaM/iaaH compared to iaaM protoplasts, whereas the level of IAA-conjugates was increased more than fivefold. For both genotypes, the location of IAA conjugates was restricted to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion to the strong cauliflower mosaic virus (CaMV) 35S promoter. Compared with the wildtype, this led to an 18-fold higher conversion of [¹⁵N₁]tryptophan to [¹⁵N₁]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protoplasts. Also in these genotypes, IAA conjugates were exclusively cytosolic. There was no major difference between transgenic and wildtype protoplasts in the proportion of chloroplastic to total cellular IAA, although the chloroplastic IAA and [¹⁵N₁IAA pools in the transformant were threefold and eightfold higher, respectively. Since the IAM pool in transgenic plants is exclusively cytosolic, these findings suggest that the increased chloroplastic [¹⁵N₁IAA pool in 35S-iaaM/iaaH protoplasts is synthesised in the cytosol but rapidly transported into the chloroplast. Furthermore, the presence of IAA in the chloroplast together with the exclusively cytosolic location of IAA conjugates, suggests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxylative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 562–568), whereas the second is located in the chloroplast and is seemingly not directly regulated inside the organelle by either of these two processes. The cytosolic IAA control mechanisms, however, also affect the chloroplastic pool size due to the rapid transport of IAA between the two compartments.