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.
Digital Object Identifier (DOI)
Banerjee, Joydeep; Sahoo, Dipak K.; Dey, Nrisingha; Houtz, Robert; and Maiti, Indu B., "An Intergenic Region Shared by At4g35985 and At4g35987 in Arabidopsis Thaliana is a Tissue Specific and Stress Inducible Bidirectional Promoter Analyzed in Transgenic Arabidopsis and Tobacco Plants" (2013). Kentucky Tobacco Research and Development Center Faculty Publications. 9.
Transient expression of P85–P87 bidirectional promoter in onion epidermal cells. A. GFP expression analysis Superimposed (bright field and green fluorescent), fluorescent and bright field images of onion epidermal cells bombarded with respective promoter construct DNA loaded gold particles are presented. Control represents untransformed onion epidermal cell visualized under CLSM. B. GUS expression analysis Light microscopy images of X-gluc treated onion epidermal cells bombarded with respective promoter construct DNA loaded gold particles are presented. Control represents untransformed onion epidermal cells treated with X-gluc.
Figure_S2.tif (7233 kB)
Confocal laser scanning microscopic analysis of empty vector control Arabidopsis seedlings at different growth stages. No detectable GFP fluorescence was observed in periphery of cotyledon in five-day old Arabidopsis seedling (A) and in leaf tip (B), apical meristemic region (C), primary root (D) and lateral root (E) of three-week-old plants. Green fluorescence image (left); bright field image (middle), superimposed image (right) are shown. Bar 250 µm in each image.
Figure_S3.tif (4376 kB)
Confocal laser scanning microscopic analysis of reproductive tissues of empty vector control Arabidopsis plants. Negligible autofluorescence in anther and stigma of flower (A) and no detectable fluorescence in the tip of silique (B) base of silique (C) are visualized. Green fluorescence image (left); bright field image (middle), superimposed image (right) are shown. Bar 250 µm in each image.
Figure_S4.tif (4309 kB)
Confocal laser scanning microscopic analysis of tobacco empty vector control plants. No detectable GFP fluorescence was observed in leaf (A) and root (B) tissues of 21-day-old tobacco seedlings. Green fluorescence image (left); bright field image (middle), superimposed image (right) are shown. Bar 250 µm in each image.
Figure_S5.tif (3668 kB)
The 1258 bp bidirectional promoter sequence located between At4g35985 and At4g35987 in head-to-head orientation. The upstream nucleotide of the start codon of At4g35985 (CAT in red) is designated as position 1 and the upstream nucleotide of the start codon of At4g35987 (ATG in red) is designated as position 1258. The major 5′-untranslated region (UTR) for At4g35985 was up to nucleotide G which is 33 bp upstream from corresponding start codon (shown as green). The TATA-box (TATAAA) was located in negative strand (brown) which is 30 bp upstream of major transcription start site (TSS) of At4g35985. The 5′-UTR for At4g35987 was up to nucleotide G which is 93 bp upstream from corresponding start codon (shown as underline) and a CAAT-box (orange) is located 74 bp upstream from TSS of At4g35987.
Figure_S6.tif (7298 kB)
Expression analysis of the Arabidopsis senescence associated gene (At4g35985) from public database. The data was collected from Genevestigator expression analysis using TAIR website (http://www.arabidopsis.org/).
Methods_S1.doc (31 kB)
Transient expression of P85–P87 bidirectional promoter in onion epidermal cells using Gene gun.