Abstract
On chromosome 4 in the Arabidopsis genome, two neighboring genes (calmodulin methyl transferase At4g35987 and senescence associated gene At4g35985) are located in a head-to-head divergent orientation sharing a putative bidirectional promoter. This 1258 bp intergenic region contains a number of environmental stress responsive and tissue specific cis-regulatory elements. Transcript analysis of At4g35985 and At4g35987 genes by quantitative real time PCR showed tissue specific and stress inducible expression profiles. We tested the bidirectional promoter-function of the intergenic region shared by the divergent genes At4g35985 and At4g35987 using two reporter genes (GFP and GUS) in both orientations in transient tobacco protoplast and Agro-infiltration assays, as well as in stably transformed transgenic Arabidopsis and tobacco plants. In transient assays with GFP and GUS reporter genes the At4g35985 promoter (P85) showed stronger expression (about 3.5 fold) compared to the At4g35987 promoter (P87). The tissue specific as well as stress responsive functional nature of the bidirectional promoter was evaluated in independent transgenic Arabidopsis and tobacco lines. Expression of P85 activity was detected in the midrib of leaves, leaf trichomes, apical meristemic regions, throughout the root, lateral roots and flowers. The expression of P87 was observed in leaf-tip, hydathodes, apical meristem, root tips, emerging lateral root tips, root stele region and in floral tissues. The bidirectional promoter in both orientations shows differential up-regulation (2.5 to 3 fold) under salt stress. Use of such regulatory elements of bidirectional promoters showing spatial and stress inducible promoter-functions in heterologous system might be an important tool for plant biotechnology and gene stacking applications.
Highlights
The intergenic region between two adjacent genes located on opposite strands of DNA is generally considered as a ‘putative bidirectional’ promoter
The intensity of GUS staining was stronger for the P85 compared to P87, (Figure 1). These results suggest that the 1258 bp intergenic region functions as a bidirectional promoter in a gene independent manner, and that the P85 promoter is relatively stronger than the P87 promoter
Bidirectional gene pairs account for a large proportion (13.3%) of all the Arabidopsis thaliana genes, confirming that this structure is prevalent in plant genomes [5]
Summary
The intergenic region between two adjacent genes located on opposite strands of DNA is generally considered as a ‘putative bidirectional’ promoter. The bioinformatic analysis and subsequent experimental studies of the available complete genome sequence of many eukaryotes (human, yeast, plants), invertebrates and vertebrates showed a genome-wide presence of bidirectional promoters [1,2,3,4,5,6,7]. From the human genome-wide analysis, the distance between two transcription start sites (TSS) of a bidirectional promoter for two adjacent divergent genes is commonly considered to be within 1000 base pairs apart [1]. Plant genomes contain intergenic region with TSS of bidirectional promoters longer than 1 kb base pairs apart [8]. Bidirectional promoters having unique regulation and expression pattern has been reported in a number of organisms including Saccharomyces cerevisiae [6,9]
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