Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras

Abstract

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.