2754 Nucleic Acids Research, 2005, Vol. 33, No. 8 each line were isolated following three outcrosses of the ori- ginal T3 plants. Homozygous plants from each of these lines required germination and initial growth on sucrose-containing medium, consistent with a defect in photosynthesis. Figure 1B compares WT and rnr1 homozygotes after 15 days of growth on MS sucrose. The cotyledons of rnr1 plants emerged either white or dark red, signifying a lack of chlorophyll and a sig- niﬁcant accumulation of anthocyanins, which dissipated after several days as the cotyledons became white and then pale green (Figure 1B, top panels). The rosette leaves of rnr1 mutants, which are already visible in rnr1-3 seedlings after 15 days, emerged and remained pale green compared with those of WT plants. Following 40 days of growth on MS sucrose, both WT and rnr1-3 were transferred to soil and were grown for an addi- tional 9 days (Figure 1B, bottom panels). Under these condi- tions, growth of rnr1-3 was signiﬁcantly slower than the WT, and rosette leaves of the mutant remained pale green. Fourteen-day-old etiolated WT and rnr1 plants were indistin- guishable, which indicated that RNR1 activity was not required for skotomorphogenesis (data not shown), and the roots of the rnr1 plants resembled those of the WT at all stages of growth. Given the strong effect of the rnr1 mutations on green tissues, it was of interest to determine whether RNR1 exhibited tissue-speciﬁc expression. RT–PCR was used to determine the mRNA abundance for RNR1 in WT seedlings, mature leaves, roots and ﬂower/ﬂower buds (Figure 1C). The highest expres- sion was found in 15-day-old seedlings, and roughly uniform expression was found in the other tissues examined, as com- pared with the control ubiquitin transcript. This result would be consistent with the presence of RNR1 in mitochondria (14,19), which are active in gene expression in non-green tissues. The extent to which RNR1 is required in plastid types other than chloroplasts is unknown. To conﬁrm the defect in chloroplast biogenesis suggested by the pale leaves of rnr1 plants, cotyledons of WT and rnr1 plants were observed by light and transmission electron micro- scopy (TEM), as shown in Figure 2. Following toluidine blue staining of transverse sections of cotyledons, chloroplasts could be detected in WT but not in rnr1 cotyledons (Figure 2A). Furthermore, rnr1 cotyledons exhibited a severely disorganized structure with no recognizable lower epithelium. Further analysis by TEM revealed that rnr1 plast- ids contained no stacked thylakoids, and that these leaves contained plastoglobules (Figure 2B), the latter perhaps reﬂecting a stress response. Taken together, these data suggest that RNR1 is essential for chloroplast development in cotyle- dons, and that this requirement is less strict in rosette leaves. mRNA and tRNA transcripts accumulate normally in rnr1 plants Because RNase II and RNase R are important for regulating RNA processing and turnover in bacteria, we compared the accumulation of representative mRNAs and tRNAs in 40-day- old WT and rnr1 plants grown on sucrose-containing medium. We observed no differences in the accumulation of mRNAs encoding the a-subunit of the CF0CF1 ATPase complex (atpA), the Photosystem II D1 protein ( psbA), the Rubisco large subunit (rbcL), or of a tricistronic mRNA encoding the PsaA/PsaB subunits of Photosystem I and the ribosomal S14 subunit ( psaA-psaB-rps14) (Figure 3A). We also observed no difference in the accumulation of the mitochon- drial mRNA cox3, which encodes cytochrome oxidase subunit III. The rnr1 mutation also had no obvious effect on 30 end processing of psbA or rbcL transcripts, a phenotype that has been observed previously in chloroplast PNPase-deﬁcient Arabidopsis (11). RNA gel blots were also probed to detect an intronless tRNA, tRNAPhe (trnF), and an intron-containing tRNA, tRNALeu (trnL). As shown in Figure 3A, we observed no differences—neither in the accumulation of tRNAPhe nor in the accumulation of the precursor or mature forms of tRNALeu. RNR1 activity is, therefore, redundant or not required for processing and turnover of chloroplast mRNA and tRNA. rnr1 plants accumulate fewer photosynthetic proteins than WT The pale leaf phenotype of the rnr1 mutants (Figure 1B) and microscopic evidence for a defect in chloroplast development (Figure 2) suggested that although chloroplast mRNAs were accumulating normally (Figure 3A), rnr1 was not Figure 2. Loss of RNR1 expression affects chloroplast biogenesis. (A) Trans- verse sections of cotyledons from 3-week-old WT and rnr1 plants stained with toluidine blue. Chloroplasts (cp) are visible in WT cotyledons as globular shapes. rnr1 cotyledons exhibit a severely disorganized structure and contain no visible chloroplasts. The magnification bar corresponds to 100 mm. (B) Transmission electron micrographs from cotyledons of 3-week-old WT and rnr1 plants. Mitochondria and plastids are indicated by m and cp, respectively. The magnification bars correspond to 1 mm.