Abstract
Nicotiana transformed with the isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens was fixed in 1% gluterdehyde and 4% formaldehyde for 1 h. Grids were treated with polygonal anti-IpTase antibody raised in rabbits and visualized with 10 nm protein-A-labeled colloidal gold. Initial localization was performed on Nicotiana transformed with the ipt gene under the control of the 35S promoter from cauliflower mosaic virus. Colloidal gold was found throughout the cell, including the cell wall, vacuole, and rough ER. Cell wall and vacuole labeling appears to be due to nonspecific binding and is greatly reduced by a BSA block. Colloidal gold label on rough ER provides preliminary evidence that translation occurs here rather than on free polysomes. General reaction throughout the cell indicates cytoplasmic activity of the enzyme. Future research will attempt to localize IPTase in wild-type Nicotiana and in plants transformed with the ipt gene under the control of the hsp 70 heat shock promoter.
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