Tissue-specific expression and promoter analysis of the tobacco Itp1 gene

Abstract

The Nicotiana tabacum Itp1 gene (Ntltp1) encodes a small basic protein that belongs to a class of putative lipid transfer proteins. These proteins transfer lipids between membranes in vitro, but their in vivo function remains hotly debated. This gene also serves as an important early marker for epidermis differentiation. We report here the analysis of the spatial and developmental activity of the Ntltp1 promoter, and we define a sequence element required for epidermis-specific expression. Transgenic plants were created containing 1346 bp of the Ntltp1 promoter fused upstream of the β-glucuronidase (GUS) gene. In the mature aerial tissues, GUS activity was detected predominantly in the epidermis, whereas in younger aerial tissues, such as the shoot apical meristem and floral meristem, GUS expression was not restricted to the tunica layer. Unexpectedly, GUS activity was also detected in young roots, particularly in the root epidermis. Furthermore, the Ntltp1 promoter displayed a tissue and developmental specific pattern of activity during germination. These results suggest that the Ntltp1 gene is highly expressed in regions of the plant that are vulnerable to pathogen attack and are thus consistent with the proposed function of lipid transfer proteins in plant defense. Deletions of the promoter from its 5′ end revealed that the 148 bp preceding the translational start site are sufficient for epidermis-specific expression. Sequence comparison identified an eight-nucleotide palindromic sequence CTAGCTAG in the leader of Ntltp1, which is conserved in a number of other ltp genes. By gel retardation analysis, the presence of specific DNA-protein complexes in this region was demonstrated. The characterization of these factors may lead to the identification of factors that control early events in epidermis differentiation.