Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. A and B, Northern blots to detect pre-rRNA processing in Nipponbare (japonica) rice under 4°C treatment for 0, 2, 4, and 6 h, with probes S7 (A) and p42 (B). Matured rRNAs stained with MB serve as the loading control. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. The relative intensities for 25S rRNA, P-A3, and 27SA2 intermediates are marked in black, red, and blue, respectively. The asterisk detected by probe S7 represents the mature 16S rRNAs. Three biological replicates were performed and a representative result is shown here. C, Northern blots to detect the 45S rRNA transcript by probe 45P in Nipponbare under 4°C treatment for 0, 2, 4, and 6 h. Both blots of 45P and p42 came from the same membrane. Matured rRNAs stained with MB serve as the loading control. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. RNA samples from two biological replicates were loaded and detected in parallel. D, Simplified model that the inhibition of rRNA biogenesis in rice by chilling stress predominantly occurs at posttranscriptional level. The 45S rRNA, transcribed by RNA Pol I from rDNAs, undergoes pre-rRNA processing to release mature rRNAs. The steady level of 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. Chilling stress inhibits pre-rRNA processing, shown by the time-course reduction of P-A3 and 27SA2 in both ITS1-first and 5′ ETS-first processing pathways, respectively (A and B). Although it remains unknown whether and how chilling treatment affect rDNA transcription, the increased 45S rRNA (C) could mainly originate from reduced pre-rRNA processing under chilling stress. The long probe 45P could distinguish the 45S rRNA from its product 35S(P).