Chloroplast RH3 DEAD box RNA helicases in maize and Arabidopsis function in splicing of specific group II introns and affect chloroplast ribosome biogenesis

Abstract

Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

Figure 1.

Phylogenetic and protein domain analyses of the DEAD box RNA helicase family. A, The RH3-containing clade (clade 7) from the phylogenetic tree of all 149 DEAD box helicases in maize, rice, and Arabidopsis and the five E. coli DEAD box RNA helicases (see Supplemental Fig. S1). Bootstrap values are indicated. B, Conserved domains of the maize, rice, and Arabidopsis DEAD box RNA helicase families. The top lines show the conserved motifs (60% minimal conservation threshold) across the 56 Arabidopsis, 46 maize, and 47 rice DEAD box RNA helicases. The bottom lines show the conserved motifs (greater than 90% identity) for the RH3 clade.

Figure 2.

RH3 proteins in maize and Arabidopsis. A, Domain organization of RH3 proteins. Two maize RH3 proteins, ZmRH3A (GRMZM2G415491_P01) and ZmRH3B (GRMZM2G163072_P01), and the AtRH3 ortholog (At5g26742) are shown. Predicted chloroplast transit peptides (TP) are shown in gray boxes. The conserved motifs in the DEAD box helicase family are indicated by black boxes and numbered (I–VI). The GUCT domain and the C-terminal G-R-S enriched domain are shaded. The region used to generate antisera is indicated. B, AtRH3 protein expresses at an early stage of development. Twenty micrograms of total proteins from green leaves at the stages indicated, or stem, flower, silique, and roots from 6-week-old Arabidopsis seedlings planted on soil, were analyzed by immunoblotting by anti-RH3 and anti-cpHSP70 antisera. The blot stained with Ponceau S is shown in the bottom panel. LHCP, Light-harvesting chlorophyll a/b-binding protein; RbcL, large subunit of Rubisco. C, ZmRH3 shows peak accumulation during the formation of chloroplasts in the sink-source transition zone of developing maize leaves. The plots are based on the identification and quantification of the RH3 homologs by mass spectrometry. For comparison, the sum of all identified plastid ribosomal proteins (53 proteins) and the sum of all identified nucleoid proteins (31 proteins) are shown. Assignment to the nucleoid and original proteomics data were from Majeran et al. (2012). NadjSPC (normalized adjusted spectral counts) is a measure of protein abundance. D, ZmRH3 proteins are localized to the chloroplast stroma, thylakoids, and nucleoids. Chloroplasts (Cp), stroma (Str), and thylakoid (Thy) were subfractionated and applied in equivalent proportions (10 μg of chlorophyll). One microgram of nucleoids (Nuc) from maize chloroplasts was applied. The blot stained with Ponceau S is shown in the bottom panel. CPN60α and D1/D2 were used as markers for the stroma and thylakoids, respectively. WHY1 is the control that localizes to stroma, thylakoids, and nucleoids.

Figure 3.

RH3 cosediments with pre-50S ribosomal subunits and interacts with specific plastid RNA. A, Maize stroma were sedimented through Suc gradients under the condition that dissociates 30S and 50S subunits. An equal proportion of each fraction was analyzed by probing an immunoblot with anti-RH3 antiserum (top panel). The same blot stained with Ponceau S is shown in the bottom panel and also visualizes the large subunit (RBCL) of the Rubisco holocomplex at 550 kD. RNA extracted from gradients was analyzed by RNA gel blotting and methylene blue staining (middle panel). 16S rRNA marks 30S ribosomal subunits, and 23S rRNA marks 50S ribosomal subunits. B, Identification of RNA ligands of RH3 using the co-IP assay. The RIP-chip assay revealed the enrichment of several RNAs (as indicated) in the RH3 co-IP pellets. C, RH3 associates with the precursor of 23S rRNA determined by poisoned-primer extension assays. Reverse transcriptase reactions were initiated with a radiolabeled primer complementary to the mature and precursor forms of 23S rRNA; a dideoxy nucleotide that terminates the reverse transcription after different distances on mature and precursor forms of 23S was included in the reactions. Pre 23S, Precursor form of 23S rRNA. To, Total; sup, supernatant; ppt, pellet.

Figure 4.

AtRH3 T-DNA insertion mutants and a double knockdown mutant with rh3-4/clpr2-1. A, T-DNA insertions in AtRH3. Exons and introns are indicated by black rectangles and lines, respectively. 5′ and 3′ untranslated regions are shown as white rectangles. The positions of the T-DNA insertions in emb1138, rh3-2 (SALK_025572), and rh3-4 (SALK_005920) are indicated by triangles. B, Embryo-lethal phenotypes associated with insertions in AtRH3. The green and mature siliques resulting from RH3-2/rh3-2 and Emb1138/emb1138 plants segregated approximately one-third white seeds and brown shriveled seeds, respectively. The shriveled seeds did not germinate. WT, Wild type. C, A rh3-4 homozygote showed a pale-green seedling phenotype, whereas the double knockdown mutant rh3-4/clpr2-1 showed an albino phenotype. Seedlings were grown for 9 d (top row) or 18 d (bottom row) on one-half-strength Murashige and Skoog medium containing 2% Suc. The rh3-4 has white cotyledons at early stages of development and exhibits pale-green true leaves. The double knockdown mutant rh3-4/clpr2-1 showed additive white seedlings. AtRH3 cDNA complemented the rh3-4 phenotype (rh3-4:RH3). Bars = 5 mm. D, RNA gel-blot analysis of RH3, CLPR2, and OEC23 mRNA levels in rh3-4, clpr2-1, and rh3-4/clpr2-1 mutants. Total RNA (6 μg) from leaf samples at leaf stage 1.07 planted on one-half-strength Murashige and Skoog medium with 2% Suc was analyzed by RNA gel blots using RH3, CLPR2, and OEC23 probes (left panels). The same blot stained with methylene blue is shown (right panels).

Figure 5.

Protein expression patterns in pale-green mutants. A, RH3, ClpR2, and cpHSP70 protein levels in rh3-4, clpr2-1, and rh3-4/clpr2-1 mutants. Total leaf proteins (30 μg of protein or dilutions as indicated) from seedlings at leaf stage 1.07 were analyzed on immunoblots by probing with anti-RH3, anti-ClpR2, and anti-cpHSP70 antisera. The Ponceau S-stained membrane at the bottom shows sample loading and abundance. LHCP, Light-harvesting chlorophyll a/b-binding protein; RBCL, large subunit of Rubisco; WT, wild type. B, Immunoblot analysis of subunits of chloroplast protein complexes in rh3-4 and clpr2-1 mutants. Total leaf extracts (20 μg of protein or dilutions as indicated) from seedlings shown at the top at leaf stage 1.07 planted on one-half-strength Murashige and Skoog medium with 2% Suc were analyzed by immunoblotting probing with antibodies for the proteins named at the right. The blot stained with Ponceau S illustrates sample loading and the abundance of the large subunits of Rubisco.

Figure 6.

Chloroplast trnA, trnI, and rps12 splicing defects in rh3-4. A, Total leaf RNA (3 μg) from wild-type (WT), rh3-4, cfm2-2, and clpr2-1 seedlings at leaf stage 1.07 was probed with trnI and trnA intron and exon probes. The blot stained with methylene blue is also shown with cytosolic and plastid rRNAs marked. B, Chloroplast rps12-int1 splicing defects in rh3-4. Poisoned-primer extension assays monitored the splicing of rps12-int1, rps12-int2, and rpl2. Total leaf RNA (10 μg) of the wild type, rh3-4, and clpr2-1 was used in reverse transcription reactions using primers mapping several nucleotides downstream of the indicated intron. O, Oligonucleotide; S, spliced; U, unspliced.

Figure 7.

Analysis of ycf3 introns 1 and 2, rpoC intron, and ClpP1 intron 1 and 2 splicing in rh3-4. Total leaf RNA (3 μg) from the wild type (WT), rh3-4, and clpr2-1 planted on one-half-strength Murashige and Skoog medium with 2% Suc until leaf stage 1.07 was analyzed by RNA gel blots (A) or by poisoned-primer extension assays for ClpP intron 1 and intron 2 in rh3-4 (B). The Arabidopsis ClpP gene contains two group II introns, but these are not seen in monocots. I, Intron; O, oligonucleotide; S, spliced; U, unspliced.

Figure 8.

Defects of chloroplast ribosome biogenesis in the rh3-4 and clpr2-1 mutants. A, Chloroplast rRNA operon in Arabidopsis and probes. The rRNA probes used in B are illustrated as white rectangles. The transcripts shown with arrows are based on Bollenbach et al. (2005). Black and dashed lines represent transcripts that were accumulated and reduced in rh3-4, respectively. B, Total leaf RNA (2 μg) from wild-type (WT), rh3-4, clpr2-1, and rh3-4/clpr2-1 seedlings at leaf stage 1.07 planted on one-half-strength Murashige and Skoog medium with 2% Suc was probed with the rRNA probes illustrated as white rectangles in A. The blot stained with methylene blue is shown to illustrate equal loading of the cytosolic rRNAs (25S and 18S). 16S and 23S* bands are plastid rRNA (23S* is a 23S rRNA fragment). p16S corresponds to the 16S precursor.

Figure 9.

RH3 protein and RNA expression patterns in clpr2-1 and the wild type during development. A, Total leaf RNA (6 μg) from the wild type (WT) and clpr2-1 at various developmental vegetative stages planted on soil was analyzed by RNA gel blot and probed with RH3 and OEC23 probes. The blot stained with methylene blue illustrates equal loading. B, Total leaf proteins (30 μg) from wild-type and clpr2-1 seedlings grown on soil at various developmental vegetative stages were tested for the accumulation of RH3 and cpHSP70. The blot stained with Ponceau S illustrates equal loading. C, Total leaf proteins (30 μg) from the wild type and various pale-green mutants (clpr2-1, ffc1-2, and tic40) grown on one-half-strength Murashige and Skoog medium with 2% Suc until leaf stage 1.07 or 1.14 were tested for the accumulation of RH3, ClpR2, and cpHSP70 using western blots. The bottom panel shows the blot stained with Ponceau S. LHCP, Light-harvesting chlorophyll a/b-binding protein; RBCL, large subunit of Rubisco.

Figure 10.

RH3 and ClpR2 do not coimmunoprecipitate with each other. Arabidopsis stroma from the wild type (WT) or clpr2-1 was used for co-IP with anti-RH3, anti-ClpR2, or anti-OEC23 antiserum. Immunoblots of coimmunoprecipitated proteins were detected by anti-RH3 or anti-ClpR2 antiserum.