We have devised a heat shock-inducible indole-3-acetic acid (IAA) synthesis system for plant cells, which is based on the iaa genes of the Agrobacterium tumefaciens T-DNA and the heat shock promoter hsp70 of Drosophila melanogaster. Two DNA constructs were tested: one contains the iaaM gene linked to the hsp70 promoter (hsp70-iaaM) and encodes the production of indoleacetamide (IAM), the other contains hsp70-iaaM and the wild-type iaaH gene which codes for the conversion of IAM into IAA (hsp70-iaaM/iaaH). Heat shock-controlled IAM and IAA synthesis was tested on two levels: biochemically by measuring IAM and IAA levels in Kalanchoe stem segments infected with the two constructs, and morphologically by IAA-dependent root formation on Kalanchoe plants, on carrot discs and on tobacco leaf fragments. At both levels the responses were found to be controlled by the heat shock promoter. IAM levels of segments infected with hsp70-iaaM increased 6-fold upon heat shock induction to 240 pmol IAM per stem segment. The accumulation of IAA in segments infected with hsp70-iaaM/iaaH and heat-shocked was found to be more variable, possibly due to IAA transport and metabolism. Heat shock treatment of Kalanchoe plants and tobacco leaf fragments infected with hsp70-iaaM/iaaH led to a strong increase in root formation. On carrot discs, heat shock-specific root induction was also demonstrated, but the responses differed between individual carrots.

The TA regions of biotype III octopine/cucumopine (OC) Ti plasmids are closely related to the TL region of the biotype I octopine Ti plasmids pTiAch5 and pTi15955. Sequence analysis shows that the limited and wide host range biotype III OC TA regions are derived from a common ancestor structure which lacked the 6a gene found in the biotype I octopine TL region. The TA region of the wide host range OC Ti plasmids has conserved most of the original TL-like structure. In most wide host range OC isolates the TA-iaaH gene is inactivated by the insertion of an IS866 element. However, the TA region of the wide host range isolate Hm1 carries an intact TA-iaaH gene. This gene encodes a biologically active product, as shown by root induction tests and indole-3-acetic acid measurements. The limited host range OC Ti plasmids pTiAB3 and pTiAg57 have shorter TA regions which are derived from a wide host range TA region. The AB3 type arose by an IS868-mediated, internal TA region deletion which removed the iaa genes and part of the ipt gene and left a copy of IS868 at the position of the deleted fragment. The pTiAB3 iaa/ipt deletion was followed by insertion of a second IS element, IS869, immediately 3' of the ipt gene. pTiAg57 underwent the same iaa-ipt deletion as pTiAB3, but lacks the IS868 and IS869 elements. Analysis of the various TA region structures provides a detailed insight into the evolution of the biotype III OC strains.

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S-beta-glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.

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