has been far less studied than upregulation of genes during abiotic stress in plants. Results Rice genome contain four genes encoding ribosomal protein rpL32 A BLASTn homology search using partial coding sequence of rpL32 in the rice genome database of NCBI (http://www.ncbi. nlm.nih.gov/) and MSU Rice Genome Annotation Project v6.1 (RGAP 6.1; http://rice.plantbiology.msu.edu/) indicated the presence of four rpL32 genes (Fig. S1). These loci have been annotated as putative 60S rpL32 gene in the NCBI database. One of these genes was present on chromosome 8 and was named rpL32_8.1. The other three were named rpL32_9.1, rpL32_9.2, and rpL32_9.3 and located contiguously on chromosome 9. The CDS and the predicted protein of all the four rpL32 members share a high percentage of identity (Fig. S2A,B). However, the 39 UTR sequence of these genes were dissimilar (Fig. S3). BLASTn homology search using 39UTR specific probes for each of the predicted rpL32 genes in NCBI database, revealed the presence of homologous expressed sequence tags (ESTs) and mRNA sequenc- es. This confirmed that all these four genes are expressed in rice. The gene structure is also similar for all the rpL32 genes of rice, each having three introns (Fig. S2C). The first intron is the smallest one and resides within the 59 UTR of the genes. The location of the remaining introns, on the other hand differs in each gene. Rice rpL32 genes are downregulated in shoots under salt stress Northern hybridization using radiolabeled 39UTR probe of rpL32_8.1 revealed that this gene is highly expressed under control (unstressed) conditions in shoots of salt tolerant indica rice Pokkali (Fig. 1A). After 1 to 12 h of salt stress, a 40–60% decrease in steady-state level of the transcript was observed relative to the unstressed condition (t0.0).The level of rpL32_8.1 decreased further to 20% of control after 24 h of salt treatment. In the salt sensitive PB1 variety of indica rice also, the level of rpL32_8.1 decreased in response to salt stress except that the response was more rapid and of greater magnitude than that observed in the salt tolerant pokkali variety (Fig. 1B). The root tissues of Pokkali and PB1 rice seedlings did not exhibit any steady decrease in their transcript level (Fig. 1C,D). rpL32_9.3 and rpL32_9.2 also exhibited downregulation of their expression in response to salt stress in shoots of Pokkali rice (Fig. 2A,B). However, the decrease in transcript level of rpL32_9.1 was relatively less under salt stress than the other three rpL32 genes (Fig. 2C). The transcript of this gene was also faintly detected in northern blots as compared to other rpL32 genes. A semiquantitative RT-PCR, using elongation factor-1a (EF-1a) as internal control gene, reconfirmed the differential downregulation of rpL32 genes in shoots of PB1 and Pokkali rice plants under salt stress (Fig. S4A–C). Though small differences in the degree of downregulation of these genes by RT-PCR and northern analyses were observed, yet the faster rate of shoot-specific downregulation of rpL32_8.1 under salt stress in PB1 rice variety was validated. Using RT-PCR, downregulation of rpL32_9.1 under salt stress was also verified. Downregulation of rpL32 genes under stress is transcriptional in origin To determine if the downregulation of rpL32 genes happens at transcriptional level, nuclear run-on assay was performed using Transcriptional Downregulation of rpL32 in Rice nuclei isolated from unstressed and stressed shoots (Fig. 3). Nuclear run-on assay of nuclei from unstressed shoots revealed that all four rpL32 genes in rice were expressed, with the strongest transcription being of rpL32_8.1 (nearly 8–10 folds higher than the other rpL32 genes; Fig. 3B,D). Although such differences might result from different hybridization efficiencies of the 39UTR specific DNA fragments used in the dot blot for the detection of rpL32 genes, it appeared least likely as these 39UTR regions are similar in length and GC content. Moreover, during RT-PCR analysis also, rpL32_8.1 was detected with lesser number of PCR cycles (than that used for other rpL32 genes). Transcription was greatly reduced in stressed shoots (treated with 200 mM NaCl for 24 h), with the signal for all the rpL32 genes being barely visible (Fig. 3C,D). This indicated that these genes are transcriptionally downregulated. As the results of this assay indicated that the majority of the rpL32 transcript in rice is contributed by rpL32_8.1, further studies were focused on the regulation of this gene. rpL32_8.1 gene expression is responsive to various treatments To investigate if rpL32_8.1 gene is responsive to other factors, its expression was studied under a variety of conditions (Fig. 4A). As compared to untreated samples, about 40% and 60–85% reduction in the transcript level was observed in response to sucrose (4.5%) and cold (4uC) treatments (for 3 and 24 h), respectively. With more than 90% reduction in the transcript level by 3 h, the most drastic effect on rpL32_8.1 expression was observed in response to drought stress (air-drying). Although some reduction in the transcript level was evident after 3 h of abscisic acid (ABA; 100 mM) treatment, such downregulation was not observed after 24 h. The transcript level of rpL32_8.1 gene was also measured in the shoots of seedlings during stress recovery (Fig. 4B). The seedlings were given 200 mM NaCl stress for 24 h, after which they were placed back onto the medium lacking NaCl. After 24 h of salt stress, expression of rpL32_8.1 was reduced as observed earlier (Fig. 4B; Lane2). However, within 1 h of stress recovery, the transcript level increased and reached the levels found in unstressed shoots (Fig. 4B; Lane 3–5). This indicates that the regulation of this gene is dynamic and reversible, and the gene is downregulated only till such time as the plants are exposed to stress. Semiquantitative RT-PCR analysis reconfirmed the decline of this transcript under cold and drought stress, and its increase during recovery from NaCl stress. (Fig. S4D–F). However, ABA treatment did not show any significant downregulation of rpL32_8.1 expression. RT-PCR showed a smaller decrease (20– 30%) in transcript levels under sucrose treatment than that observed by northern analysis. Similar to northern studies, this method also indicated that the extent of downregulation of rpL32_8.1 expression was greater in drought stress than under salt and cold stress. The transcriptional regulation of rpL32_8.1 is governed by its promoter To further confirm the transcriptional downregulation of rpL32_8.1, promoter:reporter assays were performed. A ,1.23 kb (approx), DNA fragment corresponding to the promoter region of rpL32_8.1 gene was PCR amplified using genomic DNA from Pokkali indica rice variety and cloned in TA cloning vector. The sequence of this fragment did not show any significant difference from the sequence of the same region in japonica rice, which is available at RGAP 6.1 database (Fig. 5A). An in silico search using PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e28058