The conserved endoribonuclease YbeY is required for chloroplast ribosomal RNA processing in Arabidopsis

Abstract

Maturation of chloroplast ribosomal RNAs (rRNAs) comprises several endoribonucleolytic and exoribonucleolytic processing steps. However, little is known about the specific enzymes involved and the cleavage steps they catalyze. Here, we report the functional characterization of the single Arabidopsis (Arabidopsis thaliana) gene encoding a putative YbeY endoribonuclease. AtYbeY null mutants are seedling lethal, indicating that AtYbeY function is essential for plant growth. Knockdown plants display slow growth and show pale-green leaves. Physiological and ultrastructural analyses of atybeY mutants revealed impaired photosynthesis and defective chloroplast development. Fluorescent microcopy analysis showed that, when fused with the green fluorescence protein, AtYbeY is localized in chloroplasts. Immunoblot and RNA gel-blot assays revealed that the levels of chloroplast-encoded subunits of photosynthetic complexes are reduced in atybeY mutants, but the corresponding transcripts accumulate normally. In addition, atybeY mutants display defective maturation of both the 5' and 3' ends of 16S, 23S, and 4.5S rRNAs as well as decreased accumulation of mature transcripts from the transfer RNA genes contained in the chloroplast rRNA operon. Consequently, mutant plants show a severe deficiency in ribosome biogenesis, which, in turn, results in impaired plastid translational activity. Furthermore, biochemical assays show that recombinant AtYbeY is able to cleave chloroplast rRNAs as well as messenger RNAs and transfer RNAs in vitro. Taken together, our findings indicate that AtYbeY is a chloroplast-localized endoribonuclease that is required for chloroplast rRNA processing and thus for normal growth and development.

Figure 1.

Characterization of plant and cyanobacterial YbeY proteins. A, Domain organization of YbeY proteins in photosynthetic eukaryotes, cyanobacteria, and E. coli. The UPF0054 and HAD hydrolase-like domains are shown in black and gray boxes, respectively. Plant species include Arabidopsis, Solanum tuberosum, Manihot esculenta, Populus trichocarpa, Zea mays, Oryza sativa, Physcomitrella patens, Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Synechocystis sp. PCC6803, Anabaena sp. PCC7120, Prochlorococcus marinus MIT9313, and Cyanothece sp. ATCC51142. AA, Amino acid. B, Alignment of the UPF0054 domains of YbeY proteins. The asterisks show identical amino acid residues that are conserved in all species. The conserved H3xH5xH motif is marked by black arrows and underlined. C, Semiquantitative RT-PCR analysis of AtYbeY (At2g25870) expression in Arabidopsis. The mRNA of ACTIN2 is shown as an internal control. D, qRT-PCR analysis of AtYbeY transcript levels at indicated time points after etiolated seedlings were exposed to light. Error bars indicate the sd (n = 3).

Figure 2.

Subcellular localization of the AtYbeY-GFP fusion protein in protoplasts isolated from stably transformed Arabidopsis plants. The GFP fluorescence (GFP), the chlorophyll autofluorescence (Chlorophyll), the bright-field image, and the merged images are shown. Unfused GFP and chloroplast-localized AtTHF1-GFP protein were included as controls. Bars = 5 µm.

Figure 3.

Identification of atybeY mutants and their phenotypes. A, Schematic representation of the structure of the atybeY gene and location of the T-DNA insertions in the atybeY-1 and atybeY-2 mutants (indicated by white triangles). P1 and P2 primers were used for semiquantitative RT-PCR, and P3 and P4 primers were used for qRT-PCR. B, Analysis of AtYbeY transcript levels in the wild type (WT), atybeY mutants, and AtYbeY-amiRNA lines by semiquantitative RT-PCR. C, qRT-PCR analysis of AtYbeY transcript levels in the wild type, atybeY-1, atybeY-2, the complemented line atybeY-1 (atybeY-1comp), and two AtYbeY-amiRNA lines. Error bars indicate the sd (n = 3). The expression level in the wild type was set to 100%. The mRNA of ACTIN2 is shown as an internal control. D, Phenotypes of atybeY-1, atybeY-1comp, atybeY-2, and AtYbeY-amiRNA plants grown under long-day conditions. Homozygous individuals are circled. E, Chlorophyll (Chl.) contents of the wild type, atybeY-1, and atybeY-1comp plants grown for 7 and 25 d under long-day conditions. Error bars indicate the sd (n = 5). Asterisks indicate statistically significant differences between the mutant and the wild type (**P < 0.01). fw, Fresh weight.

Figure 4.

Photosynthetic activity in leaves of wild-type (WT), atybeY-1, and complemented atybeY-1 (atybeY-1comp) plants. A, Maximum quantum efficiency of PSII (F_v/F_m). B, Light saturation curve of linear electron flux as calculated from the PSII yield (ETR II). C, Nonphotochemical quenching (qN). Error bars indicate the sd (n = 4).

Figure 5.

Loss of AtYebY expression affects chloroplast development. A, Transverse sections of cotyledons from 10-d-old wild-type (WT), atybeY-1, and complemented atybeY-1 (atybeY-1comp) seedlings stained with toluidine blue. A chloroplast in the wild-type sample is indicated by a black arrow. B, Transmission electron microscopic images of chloroplasts from cotyledons and true leaves of 10-d-old wild-type, atybeY-1, and atybeY-1comp seedlings. Bars = 100 µm (A) and 1 µm (B).

Figure 6.

Analysis of photosynthetic complexes and of representative subunits. A, BN gel electrophoretic analysis of thylakoidal protein complexes from 10-d-old wild-type (WT), atybeY-1, and complemented atybeY-1 (atybeY-1comp) seedlings. CP43, Subunit of the PSII reaction center. B, SDS-PAGE analysis of total proteins in wild-type, atybeY-1, and atbybeY-1comp seedlings. RbcL and RbcS indicate large and small subunits of Rubisco. C, Immunoblot analysis of diagnostic subunits of thylakoidal protein complexes. Isolated thylakoid samples were separated by SDS-PAGE and immunodecorated with specific antibodies against D1, D2, Cytb6, AtpB, and Lhcb1. For semiquantitative assessment, a dilution series of total protein from the wild type was loaded. A replicate Coomassie Brilliant Blue (CBB)-stained gel is shown to confirm equal loading.

Figure 7.

Expression and processing of chloroplast rRNAs in AtYbeY mutant plants. A, Structure of the chloroplast rRNA operon and location of probes used for RNA gel-blot analyses. Transcript sizes are indicated in kilobase pairs below the map. rrn16, rRNA 16S gene. B, Analysis of processing patterns of the four rRNA species encoded in the chloroplast rRNA operon. C, Accumulation of rRNAs as a proxy for the corresponding ribosomal subunits in wild-type (WT), atybeY-1, and complemented atybeY-1 (atybeY-1comp) plants. The 25S and 18S rRNAs are components of the 60S subunit and the 40S subunit, respectively, of the cytosolic ribosome. 16S and 23HB1 (largest hidden-break product of the mature 23S rRNA) are components of the 30S subunit and the 50S subunit, respectively, of the chloroplast ribosome. Error bars indicate the sd (n = 3). D, Analysis of processing patterns of three tRNAs in the chloroplast rRNA operon. Total RNAs from 10-d-old wild-type (lane 1), atybeY-1 (lane 2), and atybeY-1comp (lane 3) seedlings were separated in formaldehyde-containing agarose gels, and blots were hybridized to the probes indicated above. An ethidium bromide-stained rRNA gel is shown as a loading control.

Figure 8.

Precise 5′-end and 3′-end mapping of chloroplast rRNAs in wild-type (WT) and atybeY-1 mutant (MT) plants by cRT-PCR. PCR products were separated on ethidium bromide-stained agarose gels. The 5′ and 3′ ends of the mature rRNAs are marked by black triangles. Mapped 5′ extensions and 3′ extensions of rRNAs are shown as blue and red nucleotide (nt) sequences, respectively. The numbers at the right of the sequences represent the number of sequenced clones that contained each of the sequence variants.

Figure 9.

RNA gel-blot analysis of selected transcripts of chloroplast-encoded and nucleus-encoded genes. A, Analysis of chloroplast-encoded and nucleus-encoded mRNAs. B, Analysis of the transcripts of a subset of chloroplast-encoded trn genes. Total RNAs from 10-d-old wild-type (lane 1), atybeY-1 (lane 2), and complemented atybeY-1 (atybeY-1comp; lane 3) seedlings were separated in agarose-formaldehyde gels, and blots were hybridized to probes specific for the gene of interest. An ethidium bromine-stained gel is shown to confirm equal loading.

Figure 10.

Polysome loading of chloroplast RNAs in wild-type (WT) and atybeY-1 plants. Fractions from Suc density gradients were analyzed by RNA gel blots using gene-specific probes. Lanes 1 to 12 indicate the gradient fractions from top (15%) to bottom (55%). rrn16, rRNA 16S gene; EtBr, ethidium bromide.

Figure 11.

Endoribonuclease activity assays of AtYbeY in vitro. A, Total RNA samples (3 μg) extracted from wild-type plants were treated with AtYbeY or mutated AtYbeY (R184A and H240A) for 30 min. B, Cleavage of rbcL mRNA (250 ng) by AtYbeY or mutated AtYbeY (R184A and H240A) for 30 min. C, Degradation of trnD transcripts (250 ng) by AtYbeY or mutated AtYbeY (R184A and H240A) for 30 min. Digestion products in A to C were analyzed in agarose-formaldehyde gels. The bands of RNA ladder from top to bottom are 6.0, 4.0, 3.0, 2.0, 1.5, 1.0, 0.5, and 0.2 kb. D, Lack of degradation of double-stranded DNA (dsDNA; 200 ng) and single-stranded DNA (ssDNA; 200 ng) derived from the rbcL gene by AtYbeY for 30 min. The bands of the DNA Maker from top to bottom are 2.0, 1.0, 0.75, and 0.5 kb. E, Inhibition of AtYbeY endoribonuclease activity by the metal chelator EDTA as demonstrated by using the short synthetic oligoribonucleotide Pre16S-3* (mimicking the 3′ terminus of 16S rRNA; 300 ng) as substrate. Digestion products stained with ethidium bromide were detected in polyacrylamide gels. F and G, Analysis of in vitro endoribonuclease activity of AtYbeY using the short synthetic oligoribonucleotides Pre16S-3# (mimicking the 3′ terminus of 16S rRNA and carrying a 5′ biotin label; 250 ng) and Pre16S-5# (mimicking the 5′ terminus of 16S rRNA and carrying a 3′ biotin label; 250 ng) as substrates. Digestion products were analyzed by PAGE. The black triangles indicate cleavage sites of the synthetic oligoribonucleotide substrates (Pre16S-3*, Pre16S-3#, and Pre16S-5#) by AtYbeY in vitro, and in vivo cleavage sites of the 5′ and 3′ ends of 16S rRNA precursors are indicated by the asterisks. All assays were carried out in 50 mm HEPES-KOH (pH 7.5) buffer in a 20-μL volume at 37°C and used 2 μm of purified AtYbeY, mutated AtYbeY (R184A and H240A), or GFP.