Distinct repressing modules on the distal region of the SBP2 promoter contribute to its vascular tissue-specific expression in different vegetative organs
The Glycine max sucrose binding protein (GmSBP2) promoter directs vascular tissue-specific expression of reporter genes in transgenic tobacco. Here we showed that an SBP2-GFP fusion protein under the control of the GmSBP2 promoter accumulates in the vascular tissues of vegetative organs, which is consistent with the proposed involvement of SBP in sucrose transport-dependent physiological processes. Through gain-of-function experiments we confirmed that the tissue-specific determinants of the SBP2 promoter reside in the distal cis-regulatory domain A, CRD-A (position −2000 to −700) that is organized into a modular configuration to suppress promoter activity in tissues other than vascular tissues. The four analyzed CRD-A sub-modules, designates Frag II (−1785/−1508), Frag III (−1507/−1237), Frag IV (−1236/−971) and Frag V (−970/−700), act independently to alter the constitutive pattern of −92pSBP2-mediated GUS expression in different organs. Frag V fused to −92pSBP2-GUS restored the tissue-specific pattern of the full-length promoter in the shoot apex, but not in other organs. Likewise, Frag IV confined GUS expression to the vascular bundle of leaves, whereas Frag II mediated vascular specific expression in roots. Strong stem expression-repressing elements were located at positions −1485 to −1212, as Frag III limited GUS expression to the inner phloem. We have also mapped a procambium silencer to the consensus sequence CAGTTnCaAccACATTcCT which is located in both distal and proximal upstream modules. Fusion of either repressing element-containing module to the constitutive −92pSBP2 promoter suppresses GUS expression in the elongation zone of roots. Together our results demonstrate the unusual aspect of distal sequences negatively controlling tissue-specificity of a plant promoter.
KeywordsTranscriptional regulation Tissue-specific regulation Repressing elements Promoter distal regions Sucrose binding protein Cis-acting elements Vascular tissue-specific expression
Glycine max sucrose-binding protein 2
Vicia faba sucrose-binding protein-like protein
polymerase chain reaction
We are grateful to Dr. Simon L. Elliot for critically reading the manuscript. This research was supported by the Brazilian Government Agencies CNPq grant 50.6119/2004-1 and 470878/2006-1 (to E.P.B.F.) and FAPEMIG grant EDT 560/05 and EDT 523/07 (to E.P.B.F.). R.L.F. was supported by a graduate fellowship from the Brazilian Government Agency CAPES. C.M.C. is a FAPEMIG postdoctoral fellow (CBB 00112/07).
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