Transcriptional Downregulation of rpL32 in Rice Figure 5. Promoter sequence analysis and transcription start site (TSS) mapping of rpL32_8.1. (A) A ClustalW alignment of the japonica rice and indica rice Pokkali sequences corresponding to isolated genomic DNA portion. (B) Autoradiogram of denaturing urea polyacrylamide gel showing the three TSSs (TSS1 TSS2 and TSS3). Lanes T, A, C and G show the sequencing ladder of respective bases and the lane PE shows primer extension products. The TATA-box is marked with a shaded elliptical shape and the bands corresponding to the TSS are represented by elliptical borders. A portion of the DNA sequence downstream of TATA-Box is written besides the autoradiogram (as derived from the sequencing ladder) and the TSS bases are represented with bend arrows (showing the direction of the transcription). The distance of TSS1 (taken as +1) to the TATA-Box is also shown. (C) Schematic representation of the TATA-box and the observed TSS in the genomic DNA upstream of ATG codon of rpL32_8.1 gene. The underlined sequence represents the intron in the 59UTR portion of the gene. The shaded arrow over the sequence represents the primer used in the primer extension. ATG start codon is boldly marked. doi:10.1371/journal.pone.0028058.g005 serial deletions (DF1, DF2, DF3 and DF4). These promoter:GUS constructs were then used to raise transgenics of tobacco. Seeds collected from the GUS-positive tobacco plants were germinated on medium containing hygromycin and GUS histochemical staining was done after 2 h of transferring one-week-old seedlings to medium containing 200 mM NaCl or lacking it. The assay was performed for 3–5 different transgenic lines of each of the promoter fragments and the results are summarized with a representative picture for each transgenic (Fig. 6D–I). Plants bearing DF0 and DF1 fragments hardly showed any GUS staining in the presence or absence of salt stress (Fig. 6D,E). However, DF2 transgenic plants showed a strong GUS expression in both shoots and roots which decreased significantly in shoots upon salt treatment (Fig. 6F). DF3 transgenics also showed a strong expression of GUS but only in shoots which also decreased upon stress treatment (Fig. 6H). Plants bearing DF4 fragment showed a faint GUS expression in shoots which further decreased when subjected to salt stress (Fig. 6I). Thus, the promoter:GUS assays indicate that the transcriptional regulation of rpL32_8.1 gene originates within the promoter region of the gene. A significantly larger difference of GUS expression in unstressed shoots of DF3 and DF4 plants indicated that cis-elements required for the higher level of transcription of this gene are concentrated within a 262 bp region in the promoter (region which is present in DF3 fragment but not in DF4). Though the DF4 fragment expressed GUS faintly, it downregulated the expression of the reporter when subjected to stress. It is possible that either a repressor element is present within DF4 fragment, or the downregulation of the associated gene is caused by removal of transcription factors from the cis-elements in DF3 and DF4 fragments. For further analysis, this 262 bp DNA region was studied to determine if it was indeed the binding region of transcription factors. Downregulation of rpL32_8.1 gene is associated with removal of transcription factors DMS-LMPCR in vivo footprinting was used to investigate if any cis-element in the aforementioned 262 bp region was showing differential trans-factor binding in the presence and absence of salt. PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e28058