Figure 3. mRNA and protein accumulation in rnr1 and WT plants. (A) One microgram of total RNA from WT or from rnr1-3 was separated in a 1.2% agarose–formaldehyde gel and analyzed with the probes indicated to the right of each blot. 28S rRNA accumulation is shown in the ethidium bromide-stained membrane below each experiment to confirm equal loading. (B) Immunoblot analysis compares accumulation of the proteins shown at right in rnr1 as compared with a dilution series of WT proteins. The stained filter is shown at the bottom as an estimate of gel loading. accumulating WT levels of photosynthetic protein complexes. To test this hypothesis, total protein from WT or rnr1 leaves was analyzed by immunoblotting (Figure 3B). Because accu- mulation of photosynthetic complex subunits is highly coordinated, changes in the accumulation of individual core subunits are indicative of the accumulation of the entire complex. Results of this analysis revealed that abundance of plastid-encoded subunits of the CF0CF1 ATPase complex (AtpA), the cytochrome b6/f complex (PetA) and Rubisco (RbcL) decreased by at least 75% in the rnr1 mutant, and that the accumulation of photosystem I (PsaA/B) was nearly undetectable. Since no defect in the accumulation of the cog- nizant mRNAs had been observed (Figure 3A) (data not shown), we tentatively concluded that rnr1 mutants were defective in chloroplast translation. RNR1 is important for rRNA accumulation Because rnr1 plants appeared to have a translation defect, the accumulation and processing of chloroplast rRNAs was ana- lyzed (Figure 4). These rRNAs are encoded in a single operon that also encodes three tRNAs and is expressed as a 7.4 kb Nucleic Acids Research, 2005, Vol. 33, No. 8 2755 precursor RNA that is processed both endo- and exonucleolyt- ically (Figure 4A) (11,24,25). Blots of total leaf RNA from WT or rnr1 were probed as indicated in Figure 4B to test whether accumulation, processing or both were affected by the rnr1 mutation. When blots were probed for 16S rRNA (probe A), we found that WT leaves accumulated a mature 16S RNA transcript of 1.5 kb and low levels of a 1.7 kb precursor transcript that is generated by endonucleolytic cleavage in the 16S-tRNAIle intergenic space in the primary transcript, 180 nt down- stream of the mature 16S 30 end (6). In contrast, rnr1 plants accumulated 3-fold less mature 16S RNA than WT plants, and 3-fold more of the precursor. To positively identify the 1.7 kb RNA as the pre-16S RNA, blots were reanalyzed with probes derived from the intergenic spacer and ﬂanking the endonuc- leolytic cleavage site (probes B and C). Probe B identiﬁed the 1.7 kb RNA, while probe C did not give a signal on these blots (data not shown), conﬁrming that the longer transcript is the pre-16S RNA. When RNA gel blots were analyzed with a 23S-speciﬁc probe (probe D), a complex pattern was revealed. 23S rRNA accumulates in chloroplasts as 7 major transcripts, the most noteworthy of which are the 1.2, 1.0 and 0.5 kb 23S* bands, which represent the mature 23S transcript after processing at ‘hidden breaks’ following incorporation into ribosomes (24), and a 3.2 kb band, which represents the 23S–4.5S dicistronic precursor RNA. There were no detect- able differences in the sizes of these major 23S fragments, indicating that there were no apparent defects in 23S rRNA 30 processing, in contrast to what was observed in chloroplast PNPase-deﬁcient Arabidopsis (11). However, there were sig- niﬁcant differences in the accumulation of several 23S RNA- containing transcripts. The 7.4 kb precursor RNA was visible in the rnr1 plant, accumulation of the 3.2 kb 23S–4.5S dicis- tronic RNA increased 10-fold in rnr1, and accumulation of the 23S* fragments decreased 10-fold. A similar result was observed when blots were analyzed with a 4.5S probe (probe E): the dicistronic 23S–4.5S RNA over-accumulated in the mutant, and accumulation of the mature 0.1 kb 4.5S RNA decreased 10-fold. When RNA gel blots were analyzed for the accumulation of 5S rRNA (probe F), we found that the accumulation of mature 5S rRNA (120 nt) was at least 10-fold lower in rnr1 as com- pared with WT plants. Furthermore, mutant plants often accu- mulated low levels of an 350 nt precursor 5S rRNA (pre-5S). The fact that RNR1 activity was previously implicated in 30 exonucleolytic trimming of the mitochondrial atp9 mRNA prompted a high-resolution analysis of chloroplast rRNA 30 end processing. RNR1 activity is essential for final rRNA 30 end maturation Because the size of 16S rRNA prevents the detection of small extensions by agarose gel blot analysis, its 50 and 30 ends were mapped by cRT–PCR (Figure 5). As expected, all 16S rRNA analyzed had mature 50 and 30 ends in WT plants (ﬁlled arrow- heads). 16S rRNA 50 ends were also processed correctly in rnr1 plants, whereas no mature 16S 30 ends were detected in the mutant plants (open arrowheads). Ten of eleven clones analyzed contained a 5 nt 30 extension, and the remaining