Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase F1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where F1 coding sequence was transcribed under the control of a leaf specific Rubis co small subunit promoter (tomato RbcS-3C). Targeting the F1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistr y. The E1 produced in transgenic tobacco plants was found to be biologicall y active, and to accumulate in leaves at levels of up to 1.35% of total sol uble protein. Optimum temperature and pH for F1 enzyme activity in leaf ext racts were 81 degrees C and 5.25, respectively. F1 activity remained consta nt on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydra ted. F1 protein in old leaves, or after 5 h dehydration, was partially degr aded although F1 activity remained constant. Transgenic plants exhibited no rmal growth and developmental characteristics with photosynthetic rates sim ilar to those of untransformed SR1 tobacco plants. Results from these bioch emical and physiological analyses suggest that the chloroplast is a suitabl e cellular compartment for accumulation of the hydrolytic F1 enzyme.