three leaf stage, different treatments (dissolved in Yoshida medium) were given and tissues were collected at various time points. All experiments were done with indica rice variety Pokkali unless stated otherwise. Amplification and cloning of different genomic fragments The 39 UTR region of different rpL32 genes and the promoter fragment of rpL32_8.1 gene were amplified from the genomic DNA by two rounds of PCR (30 cycles each), the second one being done with a set of nested primers (see Table S1 and S2 for primer sequences) and cloned in TA-cloning vector (TOPO-TA, Invitro- gen). The 59 serial deletions of the promoter fragment were generated by PCR using a fixed reverse primer R1 and different forward primers (primers sequences are given in Table S3). These PCR products were cloned in pCAMBIA 1391z plant transfor- mation vector between the PstI and BamHI sites to make the promoter:GUS constructs. Northern blotting 20 mg of total RNA (isolated by using Trizol reagent from Invitrogen) was blotted onto nylon membranes according to Sambrook et al [39]. The membrane was pre-hybridized for 6 h at 55–60uC in buffer containing 5X SSC, 5X Denhardt reagent, 0.1% SDS, 100 mg/ml denatured salmon sperm DNA and 10% dextran sulphate. Thereafter, radio-labeled pre-denatured probe (generated by PCR) was added to the pre-hybridization solution. After 16–18 h of incubation at 60uC in the hybridization solution, membranes were washed once with 2X SSC at room temperature, once with 2X SSC, 0.5% SDS for 15 min and twice with 0.5X SSC, 0.5% SDS for 15 min at the hybridization temperature. The blots were imaged using a phosphorimager (Typhoon Scanner; GE Lifescience). All densitometric analyses were done using ImageJ and Microsoft excel software. Semiquantitative RT-PCR cDNA was prepared from 4 mg of total RNA using oligo dT cDNA synthesis kit (Fermentas). PCR conditions and required number of PCR cycles were standardized for each gene using 5- fold, 10-fold, 50-fold and 200-fold diluted cDNA. rpL32_8.1 was amplified using 50-fold diluted cDNA for 26 cycles. rpL32_9.2 and rpL32_9.3 were amplified using 10-fold diluted cDNA for 27 cycles. rpL32_9.1 was amplified using 5-folds diluted cDNA for 30 cycles. EF-1a was used as a control gene and was amplified using 10-fold diluted cDNA for 25 cycles. All PCRs were performed with 1 ml of the respective cDNA in 20 ml of reaction mixture. 10 ml of the amplified products were electrophoresed and imaged using gel doc. All further densitometric analyses were done using ImageJ and Microsoft excel software. The details of the primers used in RT-PCR analysis are mentioned in Table S4. Isolation of nuclei from rice shoots Nuclei were isolated by minor modification of the protocols published earlier [40,41]. Briefly, 10 g of frozen rice shoots were crushed to fine powder in liquid N2 and was homogenized with 100 ml of nuclei isolation buffer (10 mM PIPES, pH 7.0; 10 mM MgCl2; 1 M hexylene glycol; 10 mM b-mercaptoethanol; 2.5% Ficoll; 0.5% triton X-100; 2 mM spermine). After filtration through 3 layers of cheese cloth and 2 layers of miracloth (Calbiochem), and centrifugation (3300 g for 8 min), the pellet was washed thrice with washing buffer (10 mM PIPES, pH 7.0; 10 mM MgCl2; 1 M hexylene glycol; 10 mM b-mercaptoethanol), twice in presence and once in absence of triton X-100. Finally the Transcriptional Downregulation of rpL32 in Rice nuclear pellet was resuspended in 500 ml of resuspension buffer (10 mM PIPES, pH 7.0; 10 mM MgCl2; 1 M hexylene glycol; 10 mM b-mercaptoethanol; 25% glycerol), snap frozen in liquid N2 and stored at 280uC until used. DNA from the isolated nuclei were measured according to Wanner and Gruissem [42]. Nuclear run-on assay The nuclear run-on assay was done according to Hirose and Yamaya [43] and Kanazawa et al [44] using Rnasin-treated (with 100 units; Fermentas) 50 mg DNA equivalent nuclei preparation in 200 ml of 1X nuclear transcription buffer that includes 10 ml each of 10 mM ATP, GTP and CTP (Ambion) and 10 ml of a32P-UTP (3000Ci/m mol). The synthesized radiolabeled RNA was isolated in presence of 25 mg yeast t-RNA (Ambion) and equal counts of the heat denatured RNA were added to dot blot that were preincubated for 6 h at 55uC with 2 ml of hybridization buffer (250 mM sodium phosphate pH 7.2 and 100 mg/ml ssDNA). For preparing these dot blots, equal amount (500 ng) of PCR generated 39 UTR portions of different rpL32 genes and 18S rRNA, and pBSK plasmid was used. The blots were washed at 55uC in a similar way as was done for Northern hybridization and then imaged using a phosphorimager. All densitometric analyses were done using ImageJ and Microsoft excel software. Transcription start site (TSS) mapping For mapping TSS, primer extension was done with cDNA prepared from 5-10 mg of total RNA using MMLV-Reverse Transcriptase H- (Fermentas) as per manufacturer’s instruction and 50 ng of c32P end labeled primer (59-TTCGGATCGGA- GAGGGAAGC-39), designed from the 59UTR just upstream of the first intron of rpL32_8.1 gene. The primer annealing step was done at 55uC. The primer extension products were run in parallel to a marker generated by manual sequencing (Sanger’s dideoxy method manual sequencing kit; USB) of the cloned promoter fragment of rpL32_8.1 using the same primer (but unlabeled), on a 40 cm long 6% denaturing urea-PAGE gel at 60W power (,2000 V) and were compared with each other to find the TSS. Tobacco transformation via Agrobacterium Leaf discs (,1 cm2) obtained from four-week-old tobacco plants (grown axenically) were infected with secondary culture of Agrobacterium tumefaciens (A600 0.6–1.0) bearing recombinant con- structs. Shoot regeneration was done in 1X MS-Agar (including vitamins) containing 0.1 mg/L NAA and 1 mg/L BAP 250 mg/L carbenicillin, 25 mg/L hygromycin. Rooting was obtained in 1X MS-Agar (without vitamin and hormones). GUS histochemical staining and PCR with uidA specific primers was done from leaves to confirm transgenics. The seeds, collected from these T0 plants, were germinated on filter sheets soaked with 1X MS medium containing 50 mg/L hygromycin. One-week-old seedlings were treated with 200 mM NaCl solution for 2 h and GUS histochemical staining was carried out for salt treated and untreated seedlings. GUS histochemical staining assay Seedlings were first washed twice with 50 mM sodium phosphate pH 7.0. The GUS staining buffer (50 mM sodium phosphate pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1% triton X-100 and 1 mg/ml X-Gluc; Bioworld) was then vacuum infiltrated (for 10–15 min) into the samples. After this the samples were incubated in the same buffer under dark conditions at 37uC till the blue color was developed. The chlorophyll from the samples was bleached away using 70% ethanol before being photographed. PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e28058