Heat-inducible production of β-glucuronidase in tobacco hairy root cultures

Abstract

The production of beta-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36-42 degrees C for 2 h then recovery at 27 degrees C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42 degrees C heat shock and a recovery phase at 27 degrees C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl beta-D-glucuronic acid (MU) min-1 mg-1 protein at 24 h of recovery. When tissues were continuously heated at 42 degrees C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.