Hypocotyl expression and light downregulation of the soybean tubulin gene, tubB1

The tubB1 β-tubulin gene of Glycine max (previously named sβ1) is highly expressed only in rapidly elongating regions of etiolated seedling hypocotyls and this expression is strongly downregulated when the seedlings are exposed to light. Primer extension demonstrated that the gene was transcribed in these tissues and contained two sites of transcriptional initiation. To determine the mechanism regulating tubB1 expression, a chimeric reporter gene was constructed by fusing 5′ upstream regions of tubB1 to a promoterless β-glucuronidase (GUS) gene and these constructs were introduced into protoplasts by electroporation. Strong transient expression of the reporter gene was obtained after electroporation of chimeric constructs containing 1 kb of tubB1 5′ upstream sequence into tobacco protoplasts. Deletion of the distal most 300 bp from the 5′ sequence of tubB1 enhanced expression, suggesting the possibility of a negative transcriptional regulator in this region. Additional deletions of the 5′ sequence reduced expression substantially. Constructs containing a tubB1 3′ terminus were expressed at much lower levels than those containing a nopaline synthase (NOS) 3′ terminus. The tubB1-GUS chimeric gene also was introduced into tobacco by Agrobacterium-mediated Ti plasmid transformation and the organ-specific expression pattern of the chimeric gene was determined in seedlings of the transgenic plants. Hypocotyls exhibited strong GUS activity when the seedlings were germinated in darkness, but lacked the GUS enzyme when the seedlings were germinated in the light. This result demonstrates that the cis-acting elements within the first 2 kb of the translational start site of the tubB1 gene are sufficient for correct expression of the gene in etiolated, elongating hypocotyl tissues, and for the downregulation of this expression by light.