2752 Nucleic Acids Research, 2005, Vol. 33, No. 8 Arabidopsis and tomato (2,8); however, defects in ribosomal proteins do not necessarily lead to aberrant rRNA processing (9,10). None of the genes affected by the mutations discussed above encodes proteins resembling known RNA processing factors, although DCL does interact with known RNA pro- cessing enzymes (8). An apparent exception to these ﬁndings is the phosphorolytic 30 to 50 exoribonuclease, polynucleotide phosphorylase (PNPase), whose chloroplast isozyme (At3g03710) has been implicated in 23S rRNA 30 end pro- cessing (11). This leaves open the question of how chloroplast rRNA maturation is regulated, and whether this maturation is linked to other processes. The RNR superfamily of hydrolytic 30 to 50 exoribonuc- leases, which includes the prokaryotic enzymes RNase II, is, in prokaryotes, primarily involved in the turnover of mRNA and of polyadenylated RNA degradation intermediates (12). RNase R, together with PNPase, is involved in the turn- over of 16S and 23S rRNA degradation intermediates (13), and highly structured RNAs (12). The Arabidopsis nuclear genome encodes three members of the RNR superfamily, which we have named RNR1-3. Arabidopsis RNR1 (At5g02250), pre- viously named AtmtRNaseII, was ﬁrst implicated in the ﬁnal step of mitochondrial atp9 mRNA 30 end maturation (14). This enzyme was subsequently shown to also be chloroplast- targeted in Arabidopsis, and it was observed that a weak mut- ant allele of RNR1 led to the accumulation of the 23S–4.5S processing intermediate (15). Here, we demonstrate, using mutants, that RNR1 plays a profound role in rRNA metabol- ism, and thus in plant development. MATERIALS AND METHODS Plant material and growth conditions The WT ecotype Columbia (Col-0) of Arabidopsis thaliana was used in this study. Three rnr1 mutant lines, SALK_138535 (rnr1-1), SALK_044726 (rnr1-2) and SALK_090294 (rnr1-3), containing T-DNA insertions in the gene At5g02250, were obtained from the SIGnAL mutant collection (16). Mutant plants and WT controls were ger- minated and grown on MS agar (17) supplemented with 0.5% (w/v) sucrose, under ﬂuorescent lights (50 mmol·m 2 s 1) and a 16 h-light/8 h-dark photoperiod. The precise locations of the T-DNA left borders were determined by sequencing of DNA ampliﬁed by PCR from plants carrying the mutant alleles. Electron transmission microscopy analysis Cotyledon tissue samples were ﬁxed overnight in 3% glu- taraldehyde and then treated for 2 h with 10% (w/v) picric acid, 2 h with 2% uranyl acetate and stained with 0.1% (v/v) osmium tetroxide in 150 mM phosphate buffer, pH 7.2. Samples were then dehydrated through an ethanol series and inﬁltrated with EPON812 medium grade resin (Poly- sciences, Germany). Polymerization was allowed to proceed for 48 h at 60 C, after which ultrathin sections (90 mm) were cut using an Ultracut E microtome (Reichert) and col- lected on grids coated with formvar (EMS, WA). Samples were visualized with a Hitachi H-600 electron microscope operating at 75 kV. RNA isolation and RT–PCR analysis of RNR1 expression in WT and rnr1 plants RNA was extracted with Trizol (Sigma, St Louis, MO) accord- ing to the manufacturer’s instructions. For RT–PCR, RNR1 cDNA was generated from 1 mg total RNA from WT plants using AMV RT and the primer RNR30 (Table 1) according to the manufacturer’s instructions (Promega, Madison, WI). Ali- quots were added to a PCR containing the primers RNR30 and RNR50 and ampliﬁed with Takara ExTaq according to the manufacturer’s instructions (Takara Shuzo, Kyoto, Japan). PCR was performed for 28 cycles with the following program: denaturation at 95˚C for 30 s, annealing at 55 C for 30 s and extension at 72˚C for 1 min. The ubiquitin UBQ10 transcript was ampliﬁed in the same reaction as an internal control, using the primers UBQ1 and UBQ2. RNA gel blot analysis To measure chloroplast transcript accumulation, 1 mg of total RNA from WT and rnr1 plants was separated in 1.2% agarose/3% formaldehyde gels and blotted onto HyBond N+ (Amersham Biosciences, Piscataway, NJ) by capillary transfer in 25 mM phosphate buffer. Following UV-crosslinking in a Stratalinker (Stratagene, La Jolla, CA), membranes were prehybridized in Church and Gilbert buffer (18) for at least 30 min before incubation overnight with radiolabeled gene-speciﬁc probes. For high-resolution analysis, 1.5 mg total RNA from WT and rnr1 plants was analyzed using 12% (w/v) acrylamide–bisacrylamide (19:1)/7 M urea gels as described previously (19). The probes are described in the Figures 3, 4, 7, 8 and 11 and associated legends. For polysome analysis, sedimentation of crude cell lysates through 15–55% sucrose gradients was performed as described previ- ously (5,20). RNA gel blot analysis of gradient fractions was performed as described above. Table 1. Sequences of oligonucleotide primers used in this studya Primer 16-R1 cDNA 16-R2 PCR 16-F1 PCR 23-R1 cDNA 23-R2 PCR 4.5-F1 PCR 5SR1 cDNA 5SR2 PCR 5SF1 PCR T75S 5S Reverse 5S + 20 Reverse RNR30 RNR50 UBQ1 UBQ2 23S-50 23S-30 4.5S-50 4.5S-30 5S-50 5S-30 Sequence (50 to 30) GTATTAGCAGCCGTTTCCAG TCCCAAGGGCAGGTTCTTAC TAATCGCCGGTCAGCCATAC GGATTCAGCAGCAGTTCAAAAG GCATTTCGTCGCTTACTACG AGCTGAGGCATCCTAACAGAC ACCGCAGTAGAGTTTAACCACC CAAGTTCGGGATGGATTG ACGATACTGTAGGGGAGGTC T7-TATTCTGGTGTCCTAGGCGTAG ATCCTGGCGTCGAGCTATT AGGTGTTAAGCTTTTCATC AAGAAGAGCTTCAGCAGCTTCAGTG TCGATCAATATATAGCCCTCGGCCT GATCTTTGCCGGAAAACAATTGGAGGATGGT CGACTTGTCATTAGAAAGAAAGAGATAACAGG CGGTAAACGCTGGGTAGCCAAGTGCGGAGCGG T3-CCTCTTGAATTCTCAAAACTTC AAGGTCACGGCGAGACGAGCCGTT T3-ATCGAACCATGAACGAAGAAAG ATTCTGGTGTCCTAGGCGTAGAGG T3-CACCCCGTCTCCACTGGA aPositions of T3 and T7 promoter sequences are indicated in bold.