Transcriptional Downregulation of rpL32 in Rice Figure 7. DMS-LMPCR in vivo footprinting of the promoter region of rpL32_8.1. (A) In vivo footprinting of the top strand. Lane 1 shows the LMPCR products of in vitro DMS-treated piperidine fragmented genomic DNA (experimental control). Lanes 2 and 3 show the in vivo DMS-LMPCR generated products from unstressed and stressed (200 mM NaCl, 24 h) shoot samples, respectively. The distance of the respective bases from the +1 TSS is shown on the left side of the autoradiogram. The arrow heads on the right side of the autoradiogram show the sites of differential protein binding. An uncalibrated graph (drawn using ImageJ software) showing densitometric quantification of bands in each lanes is presented besides the autoradiogram. DNA sequence of the top strand corresponding to different regions of the autoradiogram has also been depicted. The underlined ‘G’ residues are the sites showing differences in the footprinting pattern and correspond to the arrow heads. (B) In vivo footprinting of the bottom strand. All representations are same as mentioned for (A)., ‘C’ residue in the top strand are marked in place of ‘G’ residues as the footprinting was done for the bottom strand. (C) Schematic representation of the cis-elements showing differential binding of transcription factors under control and stressed conditions (indicated by underlined ‘G’ and ‘C’ residues). The possible cis-elements have been represented by shaded boxes with their names. The location of TATA-Box, TSS and the third primer (R3 and F3) used in LMPCR are also shown. The number at the start of each sequence line is the base pair distance from the first TSS (+1 site). doi:10.1371/journal.pone.0028058.g007 that the transcriptional downregulation of rpL32_8.1 might be linked to the removal of transcription factors from the TCP- domain protein binding sites and other related elements. In silico analysis had also predicted the presence of GGCCC and GCCAC elements in promoters of stress downregulated genes [36]. It is being hypothesized that a similar mechanism might be mediating the decrease in transcription of other genes encoding rpL32 in rice under stress as similar cis-elements were also detected in their promoters by in silico methods. (Fig. S6). Since many ribosomal proteins are coordinately regulated, a comparison was performed between the the abundance of (A/ G)GCCCA elements within 1 kb region upstream of the ATG PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e28058