In vivo Dimethyl sulfate-Ligation mediated PCR (DMS- LMPCR) footprinting The DMS in vivo footprinting was done according to Busk and Pages [45]. Sequence of the linker primers (Linker1 and Linker2) and the primers for the blunt end generation, amplification and the final extension of top strand (R1, R2 and R3, respectively) and bottom strand (F1, F2 and F3) LMPCR are given in Table S5. For all steps of the LMPCR, Vent polymerase was used with 4 mM MgSO4 in the LMPCR buffer (20 mM Tris-HCl (pH 8.9), 40 mM NaCl, 0.009% NaCl and 0.09% Triton X-100). The amplification step was done for 18 cycles. For the final extension step, c32P- dATP end labeled primer F3 and R3 were used. After electrophoresing, the gel was dried and imaged using a phosphorimager. Sanger’s dideoxy sequencing reaction products of the cloned promoter fragment using the final extension primer was used as markers. The position of nth guanidine residue in the footprinting lane was determined as a measure of its distance from the +1 TSS by deducting 25 nucleotides from the position of coinciding base in the marker lane because the LMPCR products only contained the 25 bp linker DNA. Electrophoretic mobility shift assay (EMSA) 10 mg of nuclear proteins, prepared according to He et al [46] were incubated with c-32P end labeled double stranded oligos (50000 cpm) for 30 min in presence of 10 mM HEPES (pH 7.5), 1 mM MgCl2, 50 mM KCl, 10% glycerol, 0.5 mM DTT, 0.5 mM PMSF and 1 mg poly dI-dC in a 20 ml binding reaction at room temperature. The products were then run on a non-denaturing 4.5% polyacrylamide gel and imaged using a phosphorimager. Supporting Information Figure S1 Rice genome contains four genes encoding rpL32. One of the genes is located on chromosomes 8 (rpL32_8.1) and three others on chromosome 9 (rpL32_9.1, rpL32_9.2 and rpL32_9.3). The accession numbers as mentioned in the RGAP 6.1 database for each of these genes is shown in the figure. ORF of rpL32_9.2 and rpL32_9.3 are present on minus strand and are reverse in orientation. (TIF) Figure S2 rpL32 genes are similar in their CDS, predicted protein sequence and gene structure. (A) ClustalW alignment of the CDS of the four rpL32 genes. The top sequence represents rpL32_8.1, followed by rpL32_9.1, rpL32_9.2 and then rpL32_9.3, respectively. (B) ClustalW align- ment of the predicted protein sequence of the same. (C) Gene structure of the four rpL32 genes as represented in the RGAP 6.1 database. rpL32_9.2 and rpL32_9.3 are present in reverse orientation in the genome. Their orientation has been changed to make the comparison. For rpL32_9.1 and rpL32_9.2, two alternative spliced forms involving the first intron is predicted. The blue box represents exons. (TIF) Figure S3 ClustalW alignment of the 39UTR region of the four rpL32 genes of rice. (TIF) Figure S4 Semiquantitative RT-PCR analysis of rpL32 genes under different conditions. (A) Analysis of rpL32_8.1, rpL32_9.1, rpL32_9.2 and rpL32_9.3 expression under salt stress (200 mM NaCl, 24 h) in shoots of PB1 rice variety. (B) Expression analysis of rpL32_8.1 under salt stress in shoots of Pokkali rice variety. (C) RT-PCR analysis of rpL32_8.1 under salt stress in roots of PB1 rice variety. (D) Expression analysis of Transcriptional Downregulation of rpL32 in Rice rpL32_8.1 in shoots of Pokkali rice during stress recovery. (E) RT-PCR analysis of rpL32_8.1 under drought stress (air dried) and ABA(100 mM) treatment in shoots of pokkali rice. (F) Expression analysis of rpL32_8.1 under cold stress (4uC) and sucrose (4.5%) treatment in shoots of pokkali rice. t0 to t24 indicates the time of treatment (in h). t24-1 to t24-24 indicates the time of incubation (in h) of plants under control condition after being subjected to salt stress for 24 h. The densitometric quantification values of rpL32 genes were normalized with that for EF1-a. All analyses were performed with three technical and three biological replicates. The error bars in the histograms represent the standard deviation. Representative gel images of two biological replicates are shown in each case. (TIF) Figure S5 Important cis-elements presents in the putative promoter region of rpL32_8.1. The sequence belongs to the top strand and is presented in the 59 to 39 direction. The A residue at position 1020 represents (written in bold) the expected ATG start codon of the gene. The first intron and the 59UTR region is also shown. Different cis-elements have been provided different colors with their names mentioned below their sequence. (TIF) Figure S6 SITE II and SORLIP1 elements in the promoters of rpL32_9.1, rpL32_9.2 and rpL32_9.3. (TIF) Figure S7 Stress induced expression change and the abundance of (A/G)GCCCA elements in ribosomal protein genes. Analysis presented here is of 140 genes encoding cytoplasmic ribosomal protein having unique Affymetrix probe ID and at least two (A/G)GCCCA elements. The top and middle graph shows the fold-change in expression of genes under drought and salt stress, respectively. The lower graph shows the abundance of (A/G)GCCCA elements in the promoter of these genes. The genes have been categorized on the basis of the number of (A/G)GCCCA elements in the promoter region as represented by vertical dashed lines and the corresponding abundance of the cis-element is also written below the lower graph. The red dashed lined box in the top two graphs represents insignificant change in gene expression (,1.2 fold up- or downregulation). A lower level of fold-change in fold expression was considered as significant as this microarray was done at a early time point and also includes roots where down regulation of rpL32_8.1 was not observed. (TIF) Figure S8 Guide tree showing the presence of rpL32 in different groups of higher eukaryotes. (TIF) Figure S9 A ClustalW alignment showing similarity between rpL32 genes from different organisms. Tc- Tribolium castaneum, Dm-Drosophila melanogaster, Ag-Anopheles gambiae str. PEST, Hs-Homo sapiens, Mm-Mus musculus, Xt-Xenopus tropicalis, Os-Oryza sativa, Zm-Zea mays, At-Arabidopsis thaliana, Cr-Chlamydomonas reinhardtii, Sc-Saccharomyces cerevisiae, Sp-Schizo- saccharomyces pombe, Ss-Sclerotinia sclerotiorum, Pat-Paramecium tetra- urelia strain d4-2, Pfu-Pyrococcus furiosus DSM 3638, Tk-Thermo- coccus kodakarensis KOD1, Mk-Methanopyrus kandleri AV19. (TIF) Table S1 List of primers used for 39 UTR cloning and amplification. (DOC) PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e28058