Dithiothreitol increases b-glucuronidase accumulation in transformed tobacco (Nicotiana tabacum) protoplasts without altering their viability or the synthesis and export of cellular proteins

Abstract

The effect of dithiothreitol (DTT) on the expression of the β-glucuronidase (GUS) reporter gene under the control of the CaMV-35 S promoter has been investigated by radioactive labelling and immunoprecipitation of the enzyme in protoplasts from stably transformed tobacco plants and compared with that observed in protoplasts transiently expressing the same gene construct. An increase in net accumulation of GUS during the culture period in response to externally added DTT (2 mm) was observed both in protoplasts from transformed tobacco plants and in electroporated protoplasts. DTT had no effect on rate of degradation of the mature GUS protein, as shown in a pulse-chase experiment. Relevant aspects of protoplast physiology, such as viability, synthesis of ³⁵S-labelled cellular proteins, or synthesis and export of pathogenesis-related proteins (one putative chitinase and two 1,3-β-glucanases) were not affected by the reducing reagent.