Transcriptional Downregulation of rpL32 in Rice Figure 6. Analysis of rpL32_8.1 promoter for salt responsiveness. (A) Schematic representation depicting the length of different promoter deletion fragments upstream of ATG start codon. (B) Agarose gel image showing PCR amplification of the different promoter fragments. (C) T-DNA portion of pCAMBIA1391z. BamHI and PstI sites were used for cloning of different promoter fragments. (D) – (I) Photographs of T1 generation transgenic seedlings showing GUS histochemical staining with X-Gluc. The different conditions and construct names are mentioned in each photograph. The wild type tobacco plant was used as negative control. doi:10.1371/journal.pone.0028058.g006 A marked difference in protein binding was observed between stressed (200 mM NaCl, 24 h.) and unstressed (control) samples, at many ‘G’ residues in the top and bottom strand of the scanned region of the promoter fragment (Fig. 7A, B). In all these sites, a factor binding under the control condition (Lane 2; Fig. 7A,B) was present in reduced amounts under salt stress (Lane 3; Fig. 7A,B) which was evident from the appearance of bands in the latter case (pointed by arrowheads). LMPCR products of piperidine cleaved in vitro DMS-treated genomic DNA was used as experimental control in this assay (Lane 1; Fig. 7A,B). By superimposing ‘G’ residues detected by in vivo footprinting of top and bottom strands, over the sequence of the promoter region, three different core sequences showing differential binding of transcription factors between control and stressed samples were identified (Fig. 7C). These were 59GGCCC39 (at 2282, 2254, 2173 and 2127), 59GCCC39 (at 2240, 2209 and 2149), and 59GGGCC39 (at 2297 and 2191). Differential binding of transcription factors on 59GGGCC39 element at 2297 bp and 59GGCCC39 element at 2282 bp was detected only in top strand footprinting. It was observed that all the functional GGCCC sequences are followed by an ‘A’ residue (Fig. 7C). Similarly, all GGGCC sequences are preceded by a ‘T’ residue and GCCC sequences are flanked by an ‘A’ residue on each side. Hence, the functional cis- elements were considered as GGCCCA, TGGGCC, and AGCCCA. The sequences GGCCCA and TGGGCC are reverse complementary to each other and hence were considered as the same cis-element. Upon comparing the sequences of the remaining two cis-elements, we could assign them with a consensus sequence of (A/G)GCCCA. In PLACE database, this consensus sequence is denoted as SITE II element. The DF4 fragment, which expressed GUS very faintly and contributed to further downregulation of GUS under salt stress, was found to have a 40 bp DNA region containing a telo-box, a GCCCR type SITE II element and a SORLIP1 site. On performing an EMSA using this 40 bp DNA fragment and nuclear proteins from shoots of unstressed and stressed (200 mM NaCl, 24 h) rice seedlings, a reduction in protein binding was observed under stressed condition (Fig. 8A). Similar results were also obtained when two other oligos containing different SITE II elements, on which reduced protein binding was observed under salt stress by in vivo footprinting assays, were used in EMSA experiments (Fig. 8B,C). This substantiates the previous observa- tions and indicates that the reduction in expression of rpL32_8.1 is associated with removal of transcription factors from its promoter. Discussion The mechanism of downregulation of genes encoding ribosomal proteins under stress conditions has not been well studied. The present work indicates that the genes encoding rpL32 are transcriptionally downregulated under abiotic stress in rice and this might be mediated via removal of transcription factors from the promoter region. In silico analysis indicated that rpL32 is encoded by four genes in rice and belongs to a small multigene family as also reported earlier in plants and yeast [27–31]. The northern blots and RT- PCR analyses indicated that all rpL32 genes are downregulated under stress conditions in shoots of rice seedlings. Changes in expression pattern of various ribosomal proteins have been reported from many plant systems under different stresses [6– 8,10]. In differential cDNA libraries of barley also, this gene was found to downregulate under abiotic stress [26]. It appears that downregulation of genes encoding rpL32 under stress is a common phenomenon in monocot plants. Ribosomal proteins in plants have been reported to be translationally downregulated under osmotic stress via pathways involving TOR kinase, RAPTOR, PDK1 and S6K [12–15]. Post- transcriptional regulation of ribosomal proteins S4, S6, S28, L3, C24 (L13), L16 and P2, has also been suggested in plants [7,18– 21]. Despite identifying cis-elements and transcription factors responsible for high expression of ribosomal protein genes in growing and dividing plant cells [16,17], none of the reports describes transcriptional regulation of these genes under stress. In the current study, using nuclear run-on assay transcriptional downregulation of all four genes of rice encoding rpL32 in response to salt stress was inferred. Relatively weak detection of rpL32_9.1 in unstressed shoots during expression and nuclear run- on analyses might be due to restrictive transcription of this copy in a few specific cell types and/or at a different developmental stage, if not due to lower hybridization efficiency of the probe or the primers. In plants, variation in expression pattern among genes encoding the same ribosomal protein were also observed in previous studies [28,31]. Being the major transcript observed in our experimental conditions, the regulatory studies under salt treatment were performed for rpL32_8.1 gene. Promoter-dependent stress-responsive transcriptional downreg- ulation of rpL32_8.1 was confirmed by promoter:GUS assays in transgenic tobacco plants. The failure of DF0 and DF1 fragments to express GUS might have occurred due to the absence of an activator element in the isolated 1.2 kb promoter fragment which might have a role in suppressing the activity of a repressor element within the DF1 region. In fact, a Matrix Attachment Region was predicted 5 kb upstream of the ATG start codon of rpL32_8.1 gene. The results of promoter:GUS assays suggested that majority of the cis-elements responsible for stronger expression of rpL32_8.1 in unstressed shoots were located within the DF3 fragment; more specifically, between 284 bp and 546 bp region upstream of the ATG codon. An in vivo footprinting assay of this region indicated the presence of many (G/A)GCCCA elements where reduced binding of transcription factors was observed under salt stress. This observation was also confirmed by EMSA. (G/A)GCCCA (reverse complementary of TGGGC(C/T) sequences are variants of SITE II elements and are the binding sites of Class-I TCP-domain proteins. (G/A)GCCCA and GCCCR SITE II elements are enriched in promoters of ribosomal protein genes and have been reported to be necessary for high expression of ribosomal protein and other genes in actively growing and dividing cells of Arabidopsis and rice [16,17,32–35]. EMSA also detected reduced binding of proteins from shoots of stressed plants over a 40 bp portion of DF4 fragment containing a telo-box (TAGGGTTT), GCCCR type SITE II element (AGCCCG) and SORLIP1 site (GCCAC). This might be responsible for the decrease in GUS expression in DF4 transgenics under salt stress. Our work, hence, strongly suggests PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e28058