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A 993-bp regulatory region upstream of the translation start codon of subtilisin-like serine protease gene was isolated from  Gossypium barbadense . This (T/A)AAAG-rich region,  GbSLSP , and its 5′- and 3′-truncated versions were transferred into tobacco and Arabidopsis after fusing with  GUS  or  GFP . Histochemical and quantitative GUS analysis and confocal GFP fluorescence scanning in the transgenic plants showed that the GbSLSP-driven  GUS  and  GFP  expressed preferentially in guard cells, whereas driven by  GbSLSPF2  to  GbSLSPF4,  the 5′-truncated  GbSLSP  versions with progressively reduced Dof1 elements, both  GUS  and  GFP  expressed exclusively in guard cells, and the expression strength declined with (T/A)AAAG copy decrement. Deletion of 5′-untranslated region from  GbSLSP  markedly weakened the activity of GUS and GFP, while deletion from the strongest guard cell-specific promoter,  GbSLSPF2 , not only significantly decreased the expression strength, but also completely abolished the guard cell specificity. These results suggested both guard cell specificity and expression strength of the promoters be coordinately controlled by 5′-untranslated region and a cluster of at least 3 (T/A)AAAG elements within a region of about 100 bp relative to transcription start site. Our guard cell-specific promoters will enrich tools to manipulate gene expression in guard cells for scientific research and crop improvement.

Truncated Cotton Subtilase Promoter Directs Guard Cell-Specific Expression of Foreign Genes in Tobacco and Arabidopsis