Towards Identifying Cis and Trans Regulators of Expression of Xylem Cysteine Protease 1 (XCP1) in Arabidopsis

Abstract

Secondary xylem, commonly known as wood, is a valuable commercial commodity. Among the major components of wood are the elongated, thick-walled water-conducting cells known as tracheary elements. Understanding tracheary element differentiation and maturation is of scientific and commercial importance as it may lead to broad understanding of cellular differentiation processes as well as ways to increase both the quality and quantity of wood produced by economically important tree species. One way to begin to understand the regulation of tracheary element differentiation is to identify elements that control expression of genes associated with tracheary elements. In Arabidopsis thaliana, Xylem Cysteine Protease 1 (XCP1) is specifically expressed in tracheary elements where it catalyzes microautolysis. Thus XCP1 can serve as a useful model for identifying factors that regulate tracheary element-specific gene expression. A deletion analysis of the XCP1 promoter was conducted to identify promoter elements that are necessary and sufficient for tracheary element-restricted gene expression. Two regions required for tracheary element-specific gene expression were identified. One of these was assembled as a multimeric bait construct and used in yeast one-hybrid assays to identify candidate transcription factors that bind to the XCP1 promoter region. Subsequently, a southwestern blot analysis was used to identify transcription factors displaying specific binding to a previously reported cis-element, CTTCAAAGCCA, found in the XCP1 promoter and other tracheary element-associated genes from multiple species.