Transcriptional Downregulation of rpL32 in Rice Figure 8. EMSA with nuclear extracts from untreated and salt stressed plants. (A) EMSA with a DNA oligo corresponding to a portion of DF4 fragment containing a SORLIP1 site, a SITE II like element and a telo-box. (B) EMSA with a DNA oligo containing AGCCCA element. (C) EMSA with a DNA oligo containing GGCCCA element. Lane 1 of (A), (B) and (C) represents the experimental control lane containing no protein. Lanes 2 and 3 include nuclear extracts from untreated and stressed (200 mM NaCl, 24 h) plants, respectively. The sequence of the double stranded oligos along with their relative position in the promoter with respect to the +1 TSS is mentioned beside each autoradiogram and the cis-elements are marked. The arrows indicate the gel shift bands and the star indicates the free probes. doi:10.1371/journal.pone.0028058.g008 codon of different cytoplasmic ribosomal protein genes of rice and the level of expression of these genes under drought and salt stress. The expression data was obtained from Microarray Experiment ID GSE6901 (www.ricearray.org). Of 140 genes encoding ribosomal proteins, having a unique Affymetrix probe ID and at least two (A/G)GCCCA elements, 64.25% and 72.85% genes were found to downregulate (more than 1.2 times) under drought and salt stress, respectively. This indicates that (A/G)GCCCA elements are probably enriched in stress-downregulated genes (Fig. S7). However, the influence of other associated cis-elements on the promoter cannot be ignored as nearly 25–30% genes do not show significant change in expression under stress conditions (more than 1.2-fold up- or downregulation) and 4% of these genes were found to be upregulated significantly, even though they were highly enriched in (A/G)GCCCA elements (5 or more). Expression analysis indicated relatively moderate decrease in rpL32_8.1 transcripts in response to external sucrose treatment. No differential protein binding by in vivo footprinting was observed at the sugar-responsive elements (SRE) within the analyzed promoter fragment. In response to ABA no consistent decrease in the transcript level of rpL32_8.1 was observed. These results indicated that sugar and ABA might not be directly involved in the stable downregulation of rpL32_8.1 in rice under stress conditions. Based on the available literature and the present study, it appears that ribosomal proteins are downregulated both at transcriptional and translational level under abiotic stress condi- tions. The rate of downregulation of rpL32_8.1 transcripts seems to correlate with the severity of stress and degree of tolerance of the plant as the rate of downregulation was highest for drought stress and was higher in salt sensitive variety PB1. Gene(s) encoding rpL32 is present in all organisms ranging from archaea to higher eukaryotes and the sequence also shows high conservation among various groups of organisms (Fig. S8,S9), thereby indicating that this protein is needed for proper growth and development of all organisms. In fact in Drosophila, the deletion of this gene was found to result in various growth and developmental defects [22]. This could be the reason for its downregulation only transiently for the time for which the stress was applied and rapid return to its normal level as soon as the stress was withdrawn. rpL32_8.1 was found to get downregulated in shoots but not in roots upon exposure to salinity stress. Though the exact mechanism is not known, differential expression of genes in different tissues under various abiotic stresses is well known. The stress perception and the signaling mechanisms do vary in different tissues of plants. The prevailing environmental factors, under which plants are subjected to salinity stress, might also generate differential signals like calcium in shoots and roots [37]. Whether the rate of downregulation of such ribosomal proteins determines the stress tolerance level of a plant variety, needs further experimentation. Materials and Methods Plant material, growth conditions and sample collection Rice seedlings were grown under control conditions in growth chamber at 2862uC and 16 h photoperiod in a hydroponics culture system supplied with modified Yoshida medium [38]. At PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e28058