ANALYSIS OF 3 TISSUE-SPECIFIC ELEMENTS FROM THE WHEAT CAB-1 ENHANCER

The genes encoding the major light-harvesting antenna chlorophyll a/b- binding protein (Cab) of higher plants are regulated by light at the t ranscriptional level. In addition, their expression is largely restric ted to photosynthetically competent organs such as leaves. A 268 bp fr agment of the Cab-1 promoter from wheat functions as a light-responsiv e and organ-specific enhancer in transgenic tobacco. Using DNase I foo t-printing, four different regions have been mapped (Cab1-A, Cab1-B, C ab1-C and Cab1-D) in this fragment that bind to protein factors in tob acco nuclear extracts. Three of these regions (A, B and C) coincide wi th sequences that have been found to be functionally important from pr evious cis-element analyses. Synthetic tetramers of these three sites interact with different proteins in gel retardation assays. In additio n, cross-competition analyses demonstrate that Cab1-C is likely to int eract with ASF-2, a tobacco DNA-binding activity that binds to a conse rved GATA element found in many dicot Cab promoters. In transgenic tob acco, a 95 bp fragment of the Cab-1 enhancer containing the A, B and C regions can confer leaf expression when fused upstream of a truncated derivative of the cauliflower mosaic virus (CaMV) 35S promoter. Howev er, expression observed with this enhancer fragment in the promoter co ntext of these studies does not appear to be significantly dependent o n light. Similar results were obtained with synthetic tetramers of Cab 1-A, -B or -C. These data thus suggest that the wheat Cab-1 enhancer c ontains at least three distinct elements that contribute to leaf-speci fic expression in transgenic tobacco. Interaction between factors bind ing to these positive elements and those that bind elsewhere in the Ca b-1 enhancer may be necessary for light-responsive transcriptional act ivation.