Transcriptional Downregulation of rpL32 in Rice Figure 3. Nuclear Run-on analysis depicting the transcriptional activity of the rpL32 genes in shoots. (A) Pattern of loading of different genes in the dot blots. The blots were hybridized with 3.456106 cpm/ml probe count. (B) Blot for control (no stress) condition. (C) Blot for salt stress (200 mM NaCl, 24 h) condition. (D) Graphical representation of the results of nuclear run-on assay. Densitometric quantification values for rpL32 genes were normalized with the densitometric readings obtained for 18S rRNA. doi:10.1371/journal.pone.0028058.g003 PLACE (http://www.dna.affrc.go.jp/PLACE/) indicated the presence of various cis-elements within this region (Fig. S5). To determine the transcription start site (TSS) in this cloned fragment, a primer extension assay was carried out using total RNA from shoots. The result indicated the presence of three major TSSs which were named as TSS1, TSS2 and TSS3 (Fig. 5B,C). The TSS1 was considered as the +1 TSS. No conventional TATA-box was found in the vicinity of the TSSs, but a TATA-like sequence TATAAGA was found 27 bp upstream of the +1 TSS (Fig. 5B,C). To identify the minimum functional promoter region respon- sible for the expression and stress responsiveness of rpL32_8.1, promoter:GUS constructs were prepared in pCAMBIA 1391z vector (Fig. 6A–C) using a 1170 bp DNA fragment (DF0; considered as the full length promoter fragment) and its four Figure 4. Northern blot analysis of rpL32_8.1 under various treatments and during stress recovery. (A) Expression analysis under various treatments (sucrose, cold, drought and ABA) as mentioned below each lane. t0.0 to t24.0 indicate the time of treatment (in h). (B) Transcript abundance during stress recovery. t0 (lane 1) and t24 (lane 2) indicate the control (unstressed) and 24 h stressed (200 mM NaCl) tissues. t24-1 (lane 3), t24-15 (lane 4) and t24-24 (lane 5) indicate 1, 15 and 24 h of incubation during recovery from salt stress. All analyses were done as mentioned for Figure 1. doi:10.1371/journal.pone.0028058.g004 PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e28058