TRANSGENIC TOBACCO PLANTS EXPRESSING THE ARABIDOPSIS-THALIANA NITRILASE-II ENZYME

Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts ind ole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid i n vitro. To probe the capacity of this enzyme under physiological cond itions in vivo, the cDNA PM255, encoding nitrilase II, was stably inte grated into the genome of Nicotiana tabacum by direct protoplast trans formation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, bas ed on the occurrence of its mRNA and polypeptide. The enzyme was catal ytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg(-1) protein with indole-3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[C-13]-indole-3-acetoni trile to 1-[C-13]-indole-3-acetic acid in vivo as determined by HPLC/ GC-MS analysis. Untransformed tobacco was unable to catalyze this reac tion. When transgenic seeds were grown on medium in the absence of ind ole-3-acetonitrile, germination and seedling growth appeared normal. I n the presence of micromolar levels of exogenous indole-3-acetonitrile , a strong auxin-overproducing phenotype developed resulting in increa sed lateral root formation (at 10 mu M indole-3-acetonitrile) or stunt ed shoot growth, excessive lateral root initiation, inhibition of root outgrowth and callus formation at the root/shoot interface (at 100 mu M indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitr ile to indole-3 acetic acid in vivo, even when expressed at subphysiol ogical levels thereby conferring a high-auxin phenotype upon transgeni c plants. Thus, the A. thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirement s for auxin biosynthesis in vivo.