COMPARTMENTATION OF INDOLE-3-ACETIC-ACID METABOLISM IN PROTOPLASTS ISOLATED FROM LEAVES OF WILD-TYPE AND IAA-OVERPRODUCING TRANSGENIC TOBACCO PLANTS

Cellular compartmentation of indole-3-acetamide (IAM), indole-3-acetic acid (IAA), and [N-15(1)]IAA synthesised from [N-15(1)]tryptophan was monitored in protoplasts isolated from sterile wild-type tobacco SR1 plants, and in IAA-overproducing plants expressing the Agrobacterium t umefaciens T-DNA IAA genes iaaM and iaaH. Indole-3-acetamide was locat ed exclusively in the cytosol of both iaaM and iaaM/iaaH protoplasts, being 75% lower than in iaaM protoplasts, presumably because of conver sion into IAA by action of the iaaH-encoded hydrolase. The free-IAA le vel, however, was raised only 8% in iaaM/iaaH compared to iaaM protopl asts, whereas the level of IAA-conjugates was increased more than five fold. For both genotypes, the location of IAA conjugates was restricte d to the cytosol, while one-third of the free-IAA pool was present in chloroplasts. Transcription of the iaaM gene was increased by fusion t o the strong cautiflower mosaic virus (CaMV) 35S promoter. Compared wi th the wildtype, this led to an 18-fold higher conversion of [N-15(1)] tryptophan to [N-15(1)]IAA, a three- to fourfold increase in free IAA, and a tenfold higher level of IAA conjugates in 35S-iaaM/iaaH protopl asts. Also in these genotypes, IAA conjugates were exclusively cytosol ic. There was no major difference between transgenic and wildtype prot oplasts in the proportion of chloroplastic to total cellular IAA, alth ough the chloroplastic IAA and [N-15(1)]IAA 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 [N-15(1)]IAA pool in 35S-iaaM/iaaH pr otoplasts is synthesised in the cytosol but rapidly transported into t he chloroplast. Furthermore, the presence of IAA in the chloroplast to gether with the exclusively cytosolic location of IAA conjugates, sugg ests the presence of two differentially subcellular pools of IAA. The first is located in the cytosol and mainly regulated by non-decarboxyl ative catabolism and conjugation (Sandberg et al. 1990, Planta 180, 56 2-568), whereas the second is located in the chloroplast and is seemin gly 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.