Plant Physiology and Biochemistry 82 (2014) 309e318

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Research article

A chloroplast-localized DEAD-box RNA helicaseAtRH3 is essential for intron splicing and plays an important role in the growth and stress response in Arabidopsis thaliana Lili Gu a, Tao Xu a, Kwanuk Lee a, Kwang Ho Lee b, Hunseung Kang a, * a

Department of Plant Biotechnology and Landscape Architecture, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea Department of Wood Science and Landscape Architecture, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 June 2014 Accepted 3 July 2014 Available online 10 July 2014

Although many DEAD-box RNA helicases (RHs) are targeted to chloroplasts, the functional roles of the majority of RHs are still unknown. Recently, the chloroplast-localized Arabidopsis thaliana AtRH3 has been demonstrated to play important roles in intron splicing, ribosome biogenesis, and seedling growth. To further understand the functional role of AtRH3 in intron splicing and growth and the stress response in Arabidopsis, the newly-generated artificial microRNA-mediated knockdown plants as well as the previously characterized T-DNA tagged rh3-4 mutant were analyzed under normal and stress conditions. The rh3 mutants displayed retarded growth and pale-green phenotypes, and the growth of mutant plants was inhibited severely under salt or cold stress but marginally under dehydration stress conditions. Splicing of several intron-containing chloroplast genes was defective in the mutant plants. Importantly, splicing of ndhA and ndhB genes was severely inhibited in the mutant plants compared with the wildtype plants under salt or cold stress but not under dehydration stress conditions. Moreover, AtRH3 complemented the growth-defect phenotype of the RNA chaperone-deficient Escherichia coli mutant and had the ability to disrupt RNA and DNA base pairs, indicating that AtRH3 possesses RNA chaperone activity. Taken together, these results demonstrate that AtRH3 plays a prominent role in the growth and stress response of Arabidopsis, and suggest that proper splicing of introns governed by RNA chaperone activity of AtRH3 is crucial for chloroplast function and the growth and stress response of plants. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Abiotic stress Arabidopsis thaliana Chloroplast DEAD-box RNA helicase Intron splicing RNA chaperone

1. Introduction Chloroplasts possess less than 150 genes that encode messenger RNAs, ribosomal RNAs, and transfer RNAs in its own circular genome, but recent analyses of genome sequences and the proteins localized in specific cellular organelles of plants have demonstrated that thousands of nuclear-encoded proteins are targeted to the chloroplast and are involved in chloroplast gene expression, biogenesis, and function (Peltier et al., 2006; Olinares et al., 2010; Stern et al., 2010). Chloroplast gene expression is commonly regulated during the process of posttranscriptional RNA

Abbreviations: RBP, RNA-binding protein; RH, DEAD-box RNA helicase. * Corresponding author. Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea. Tel.: þ82 62 530 2181; fax: þ82 62 530 2069. E-mail address: [email protected] (H. Kang). http://dx.doi.org/10.1016/j.plaphy.2014.07.006 0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.

metabolism, including mRNA processing, splicing, editing, decay, and translation (del Campo, 2009; Stern et al., 2010). Chloroplast RNA metabolism involves several complicated processes that have both prokaryotic and eukaryotic characteristics (SchmitzLinneweber and Barkan, 2007). The splicing of group I and group II introns is one particular example in which, instead of selfsplicing, intron splicing in chloroplasts requires many nuclearencoded proteins (Jenkins et al., 1997; Asakura and Barkan, 2006, 2007; Barkan et al., 2007). A variety of nuclear-encoded RNAbinding proteins (RBPs) are targeted to chloroplasts and play indispensible roles in posttranscriptional regulation of RNA metabolism and gene expression in chloroplasts (del Campo, 2009; Schmitz-Linneweber and Small, 2008; Stern et al., 2010). RNA helicases are ubiquitous proteins that catalyze the unwinding of duplex RNA secondary structures in an ATP-dependent manner. DEAD-box RNA helicases (RHs) belong to the super family 2 (SF2) among the six SFs of RHs (Tanner and Linder, 2001; Singleton

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et al., 2007). Genome sequence analyses have revealed that plants harbor multiple RH family members. For example, the Arabidopsis thaliana genome contains 58 DEAD-box RHs and the rice (Oryza sativa) genome harbors more than 50 DEAD-box RHs (Mingam et al., 2004; Umate et al., 2010). RNA helicases participate in the regulation of essentially all cellular processes involving RNAs, including transcription, pre-mRNA splicing, ribosome biogenesis, mRNA transport, translation initiation, RNA decay, and organelle gene expression (Rocak and Linder, 2004; Linder and Jankowsky, 2011). The roles of RHs in the metabolism of aberrant and silencing RNAs during the regulation of mRNA quality control and gene expression in plant development have been demonstrated (reviewed in Linder and Owttrim, 2009). The functional roles of several RHs have also been demonstrated in the stress responses of plants (Gong et al., 2005; Kant et al., 2007; Kim et al., 2008; Li et al., 2008). Although the nuclear and cytoplasmic cellular function of a number of RHs have been determined, reports addressing the roles of RHs in chloroplast gene expression and function are severely limited. Several Arabidopsis DEAD-box RHs, including AtRH3, AtRH22, AtRH26, AtRH39, AtRH47, AtRH50, and AtRH58, have been determined to be localized to chloroplasts (Peltier et al., 2006; Zybailov et al., 2008; Olinares et al., 2010). It has been demonstrated that AtRH39 is involved in post-maturation processing of chloroplast 23S rRNA (Nishimura et al., 2010), and that AtRH22 functions in the assembly of 50S ribosomal subunits in chloroplasts (Chi et al., 2012). It has been reported recentlythatAtRH3 null mutants are embryo lethal, and a weak allele (rh3-4) with a T-DNA inserted at near the C-terminal end (approximately 100 base pairs upstream of the stop codon) of AtRH3 results in pale-green seedlings with defects in the splicing of specific group II introns and chloroplast ribosome biogenesis (Asakura et al., 2012) and shows abnormal chloroplast biogenesis and ABA-deficient and NaClsensitive phenotypes (Lee et al., 2013). To further understand the functional role of AtRH3 in intron splicing and stress response in Arabidopsis, we generated and analyzed artificial microRNAmediated knockdown (amiR) plants as well as the T-DNA tagged rh3-4 mutant under abiotic stresses, including cold, salt, and dehydration. We provide evidence demonstrating that proper splicing of introns governed by AtRH3 RNA chaperone activity is crucial for the growth and stress response of the plant. 2. Materials and methods

Tables 1 and 2, respectively. The pBI121 vector expressing amiRNA under the control of cauliflower mosaic virus 35S promoter was introduced into Arabidopsis by vacuum infiltration using Agrobacterium tumefaciens GV3101. The T3 or T4 homozygous lines were selected and used for phenotype investigation. 2.3. RNA extraction, RT-PCR, and Northern blot analysis Total RNA was extracted from frozen samples using the Plant RNeasy Extraction kit (Qiagen, Valencia, CA, USA), treated with RQ1 DNase (Promega, Madison, WI, USA), and further purified using an RNeasy Clean-up kit (Qiagen). Two hundred nanograms of RNA were reverse transcribed and amplified using a One-step RT-PCR kit (Qiagen) with the gene-specific primers listed in Supplemental Table 3. To analyze the splicing patterns of intron-containing genes by Northern analysis, 6 mg of total RNA were separated on a 1% agarose gel and transferred to a hybond-NX membrane (Amersham Biosciences, Piscataway, NJ, USA). RNA blots were hybridized in 1 x Ultrahyb-Oligo hybridization buffer (Ambion, Grand Island, NY, USA) with a radiolabeled DNA probe, which was labeled by [a-32P] dCTP using random primer DNA labeling kit (TAKARA BIO INC., Tokyo, Japan) or by [g-32P] ATP using T4 polynucleotide kinase end-labeling kit (TAKARA BIO INC.). After washing the membrane with washing buffer (2 x SSC, 0.1% SDS) for 10 min at room temperature and with washing buffer (0.25 x SSC, 0.1% SDS) for 30 min at 42  C, the signals for spliced products were detected using a Phosphorimager FLA7000 (GE health, Pittsburgh, PA, USA). 2.4. Analysis of splicing efficiency by real-time RT-PCR Splicing efficiency of the chloroplast transcripts in the wild-type and mutant plants was analyzed by quantitative real-time RT-PCR as described (Koprivova et al., 2010; Cohen et al., 2014). Gene-specific oligonucleotide primers corresponding to the exon/exon regions were designed to amplify mature (spliced) transcripts, and the primers corresponding to the intron/exon regions were designed to amplify precursor (unspliced) transcripts (Supplemental Fig. S1). Two hundred nanograms of RNA were reverse transcribed and amplified in a Rotor-Gene Q real-time thermal cycling system (Qiagen) using QuantiTect SYBR Green RT-PCR kit (Qiagen) with the gene-specific primers listed in Supplemental Table 4. The ratios of spliced mature transcripts and unspliced precursor transcripts were calculated between the wild-type and mutant plants.

2.1. Plant materials and growth conditions 2.5. Chlorophyll content and chlorophyll fluorescence measurement Arabidopsis thaliana Columbia-0 ecotype (Col-0) was grown either on half-strength Murashige and Skoog (MS) medium containing 1% sucrose or in soil at 23  C under long-day conditions (16h-light/8-h-dark cycle). The Arabidopsis T-DNA mutant (SALK_005920) with a T-DNA insertion in the AtRH3 gene (rh3-4) was obtained from the Arabidopsis Biological Resource Center. To evaluate the response of plants to abiotic stresses, seeds of wildtype and mutant plants were sown on half-strength MS medium supplemented with 75e200 mM NaCl or 100e300 mM mannitol. Seed germination and seedling growth were measured on the indicated days, essentially as described previously (Kim et al., 2008).

Chlorophyll content was measured using the 80% acetone extraction method as described previously (Inskeep and Bloom, 1985). Three-week-old wild-type and mutant plants were ground in liquid nitrogen, and the chlorophyll was extracted with 80% acetone. After centrifugation of the extracts at 10,000 rpm for 10min, the absorbance of the supernatant was measured at 664 nm and 647 nm. Chlorophyll fluorescence (Fv/Fm) was measured with a Handy PEA chlorophyll fluorimeter according to the manufacturer's instructions (Hansatech Instruments Ltd., Norfolk, UK). 2.6. Transmission electron microscopy

2.2. Construction of knockdown mutants using artificial microRNA To construct AtRH3 knockdown mutants via an artificial microRNA (amiRNA)-mediated knockdown strategy (Schwab et al., 2006), three different amiRNAs were generated using WMD3-Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/ webapp.cgi). The AtRH3 target sites of each amiRNA and the primers used for amiRNA construction are listed in Supplemental

Two-week-old Arabidopsis seedlings were fixed with a mixture of 2% glutaraldehyde (v/v) and 2% paraformaldehyde (v/v) in 0.05 M cacodylate buffer, pH 7.2, at room temperature for 4 h. After washing the sample in the same buffer, the samples were post-fixed with 1% osmium tetroxide in 0.05 M cacodylate buffer at room temperature for 1 h. The fixed samples were washed in buffer and then dehydrated in an ethanol series of 30e100%. The samples were embedded

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in LR White (London Resin Co., London, UK) at 50  C for 24 h, and ultrathin sections (80e100 nm thickness) were prepared using an ultra microtome with a diamond knife. The thin sections were stained with uranyl acetate and lead citrate and then examined using a transmission electron microscope JEM-1400 (Jeol, Tokyo, Japan). 2.7. Analysis of RNA chaperone activity The AtRH3 coding region was cloned into the NdeI/BamHI site of the pINIII vector, and cold shock assay in Escherichia coli BX04 mutant cells (Xia et al., 2001) was conducted essentially as described previously (Kim et al., 2007). The E. coli cells transformed with the pINIII vector were grown in LuriaeBertani (LB) medium to an optical density of 0.8 at 600 nm, and the cell cultures were subjected to serial dilution, spotted on LB-agar plates containing IPTG, and incubated at low temperatures. For the transcription antitermination assay, E. coli RL211 cells (Landick et al., 1990) were transformed with each construct, grown in liquid LB medium, and spotted on LB-carbenicillin plates with or without chloramphenicol. The molecular beacon, a 78-nucleotide-long hairpin-shaped DNA molecule labeled with a fluorophore (tetramethylrhodamine) and quencher (dabcyl) (Kim et al., 2007), was synthesized for the in vitro nucleic acid-melting assay, and a DNA melting assay was conducted essentially as described previously (Kim et al., 2007). The recombinant GST-AtRH3 fusion proteins were purified from E. coli and reacted with the molecular beacon, and the emitted fluorescence was measured using a Synergy H1 Hybrid Reader (BioTek, Winooski, VT, USA) at excitation and emission wavelengths of 550 nm and 580 nm, respectively. 3. Results 3.1. AtRH3 mutants display retarded growth under normal growth conditions The homozygous T-DNA insertion mutant rh3-4 (SALK005920) analyzed here has a T-DNA insertion in intron 9, which is approximately 100 base pairs upstream of the stop codon (Fig. 1A). It has been previously reported that rh3-4 mutant displays the pale-green and delayed-growth phenotypes under normal growth conditions (Asakura et al., 2012) and shows abnormal chloroplast biogenesis and ABA-deficient and NaCl-sensitive phenotypes (Lee et al., 2013). To further confirm the pale-green and delayed-growth phenotypes observed in homozygous rh3-4 mutant, we generated amiRNAmediated knockdown plants. Among the three amiRNA transgenic plants targeted to the different sites of the AtRH3 gene (Supplemental Table 1), amiRNA1 (amiR1) showed obvious phenotypes and was selected for further analysis. The levels of AtRH3 in amiR1 mutants decreased to half of that in the wild-type plant (Fig. 1B). Both rh3-4 and amiR1 mutants displayed pale-green and

Fig. 1. Retarded-growth phenotypes and defects in chloroplast biogenesis and photosynthetic activity in rh3-4 mutant plants. (A) Schematic presentation of T-DNA insertion site and amiRNA (amiR) target site forAtRH3. Black rectangles and lines represent exons and introns, respectively, and 50 - and 30 - untranslated regions are indicated by white rectangles. The positions of T-DNA insertion in the rh3-4 and amiR target site are indicated by triangles. (B) RT-PCR analysis to confirm the absence and downregulation of AtRH3 expression in rh3-4 mutant and two amiR transgenic lines (amiR1-1 and amiR1-2). The amplification of tubulin transcripts in the same RNA samples is shown as a control. (C) Retarded growth and development of rh3-4mutant and amiR lines. (D) The wild type (Col-0) and rh3-4 mutant plants were grown in soil, and the number of leaves at the time of bolting was measured. (E) Chloroplast structures of 2-week-old wild type (Col-0) and amiR1-1 mutant were observed by transmission electron microscopy. Bars ¼ 1 mm. (F) Chlorophyll fluorescence (Fv/Fm) of 3-week-old leaves was measured using a Handy PEA chlorophyll fluorimeter. Data are mean ± SE obtained from five independent experiments.

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delayed-growth phenotypes (Fig. 1C). Notably, the degree of severity of pale-green and delayed-growth phenotypes was correlated with the levels of AtRH3 expression in that rh3-4 knockout mutant showed much severe phenotypes than amiR1 knockdown mutant plants. Bolting of the mutants was delayed for approximately 1 week compared with that of the wild-type plants. However, the number of leaves at the time of bolting was similar between the wild-type and mutant plants (Fig. 1D), suggesting that AtRH3 does not influence the control of flowering time. Our present data and the previous reports clearly demonstrate that AtRH3 is essential for normal growth of Arabidopsis plants. 3.2. AtRH3 is essential for chloroplast development and function A previous report showed that rh3-4 mutant displayed abnormal chloroplast structures (Lee et al., 2013). To further support the previous findings that AtRH3 is essential for normal chloroplast development and function, we analyzed by TEM chloroplast structure in the amiR1 mutant lines as well as in rh3-4 mutant. The grana lamellae of the thylakoid membrane in the wild-

type plants was intact and regular, whereas the amiR1 mutants had fewer stacked thylakoids and exhibited a disturbed thylakoid membrane organization (Fig. 1E and Supplemental Fig. S2A). It was noted that the defect in thylakoid structure in the amiR1 knockdown mutant lines was less severe than that in the rh3-4 knockout mutant (Supplemental Fig. S2A). These results indicate that AtRH3 is necessary for the formation of normal chloroplast structures. We next measured chlorophyll fluorescence and chlorophyll content in the wild-type and mutant plants. The ratio of variable fluorescence (Fv) over the maximum fluorescence value (Fv/Fm), a standard measure of photosystem II integrity, was measured in 3week-old wild-type and mutant plants. The Fv/Fm of the wildtype plants was approximately 0.85, which is normal for the wild-type plants, whereas the Fv/Fm values of the rh3-4 mutant and amiR1 lines were approximately 0.62 and 0.80, respectively (Fig. 1F). The pale-green phenotype of amiR1 plants was much clearer at younger stages, and the Fv/Fm value of amiR1 lines was much lower (approximately 0.7) when measured at 12-day-old seedlings. Total chlorophyll contents in the rh3-4 and amiR1 mutants decreased to 73e78% of the wild-type plants (Supplemental

Fig. 2. Effect of dehydration stress on seedling growth of rh3-4 mutant plants. The wild type (Col-0), rh3-4 mutant, and amiRNA transgenic lines (amiR1-1 and amiR1-2) were grown on MS medium or MS medium supplemented with 150 mM mannitol, and the photographs were taken 10 days and 18 days after germination. Percentage of green leaves in total seedlings was calculated, and data are mean ± SE obtained from three independent experiments.

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Fig. S2B). The levels of chlorophyll a were reduced in the rh3-4 and amiR1 mutants compared with the wild-type plants but chlorophyll b levels remained constant in the wild-type and mutant plants (Supplemental Fig. S2B). These results show that decreased AtRH3 expression causes a deficiency in chloroplast biogenesis and photosynthetic efficiency, suggesting that AtRH3 is crucial for normal chloroplast development and function. 3.3. AtRH3 mutants are sensitive to cold or high salt stress A recent report demonstrated that rh3-4 mutant was more sensitive to salt stress than the wild-type plants (Lee et al., 2013). To determine whether AtRH3 plays a role in plant response to other environmental stresses, the growth and stress response of the wildtype and mutant plants were analyzed under abiotic stress conditions, including cold, dehydration, and high salt stresses. Although the growth of the mutants was delayed compared with that of the wild-type plants 10 days after germination on normal MS medium, the growth of the rh3-4 and amiR1 mutant seedlings was comparable with that of the wild-type seedlings 18 days after germination (Fig. 2). When the plants were grown on MS medium supplemented

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with 150 mM mannitol for 18 days, the size of the mutant seedlings was comparable with that of the wild-type seedlings and greening of the leaves of the mutant seedlings was marginally inhibited compared with that of the wild-type seedlings (Fig. 2). However, when the plants were grown on MS medium supplemented with 100 mM NaCl, it was evident that the growth and leaf greening of both the rh3-4 and amiR1 mutant seedlings was significantly inhibited compared with those of the wild-type seedlings (Fig. 3), which is consistent with the previous results demonstrated by the analysis of rh3-4 mutant (Lee et al., 2013). In addition, cold stress greatly affected the growth of the plants in that the growth and leaf greening of both rh3-4 and amiR1 mutant seedlings was significantly inhibited compared with those of the wild-type seedlings at low temperatures (Fig. 3). Notably, the degree of severity of growth inhibition and pale-green phenotypes was correlated with the levels of AtRH3 expression in that rh3-4 knockout mutant showed much severe phenotype than amiR1 knockdown mutant plants. Root length of the rh3-4 and amiR1 mutant plants was reduced compared with that of the wild-type plants grown on normal MS medium (Supplemental Fig. S3). Although root length of the wild-type and mutant plants decreased under stress conditions, these stresses did

Fig. 3. Effect of cold or salt stress on seedling growth of rh3-4 mutant plants. The wild type (Col-0), rh3-4 mutant, and amiRNA transgenic lines (amiR1-1 and amiR1-2) were grown on MS medium at 10  C for cold stress and on MS medium supplemented with 100 mM NaCl for salt stress, and the photographs were taken at the indicated days after germination. Percentage of green leaves in total seedlings was calculated, and data are mean ± SE obtained from three independent experiments.

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not cause a specific reduction in root length of the mutant plants under the stress conditions (Supplemental Fig. S3). All of these results show that AtRH3 affects Arabidopsis seedling growth significantly under cold or salt stress conditions but marginally under dehydration stress conditions. 3.4. AtRH3 is crucial for correct splicing of chloroplast introns A previous report demonstrated that AtRH3 is involved in the splicing of some chloroplast intron-containing genes, including rps12, rpl21, rpl22, trnI, and trnA, in Arabidopsis (Asakura et al., 2012). To further determine whether AtRH3 affects the splicing of other intron-containing chloroplast genes, the splicing patterns of all intron-containing genes (16 mRNAs and 6 tRNAs) in Arabidopsis chloroplasts were analyzed by RT-PCR, Northern blotting, and quantitative real-time RT-PCR. Our RT-PCR analysis clearly supported the previous findings that AtRH3 affects splicing of rps12, rpl21, rpl22, trnI, and trnA. In addition, RT-PCR and Northern blotting analysis showed that AtRH3 affects the splicing of atpF, trnL, and trnK (Fig. 4). Quantitative real-time RT-PCR analysis further revealed that the ratios of spliced and unspliced atpF, trnL, and trnK transcripts were significantly lower in rh3-4 and amiRNA mutants than in the wild-type plant (Fig. 4C), suggesting that AtRH3 affects the splicing of atpF, trnL, and trnK genes. Interestingly, the degree of splicing defects was correlated with the severity of the phenotypes in that the ratios of spliced and unspliced atpF, trnL, and trnK transcripts were much lower in rh3-4 mutant that showed severe phenotypes than in amiR1 mutants that showed weak phenotypes (Figs. 3 and 4). These results and previous findings indicate that AtRH3 affects splicing of many intron-containing genes in chloroplasts, which is crucial for normal seedling growth. Because rh3-4 and amiR1 mutant plants are sensitive to cold and salt stress (Fig.3), it is interesting to determine whether the AtRH3mediated splicing of chloroplast genes is affected by specific abiotic stresses. To address this question, the splicing patterns of all introncontaining chloroplast genes were analyzed in the wild-type and mutant plants under stress conditions. The results showed that splicing of most chloroplast genes was not altered in the mutant plants under stress conditions. However, splicing of two genes, ndhA and ndhB, was significantly affected by stress treatment (Fig. 5). Both the precursor and mature forms of ndhA and ndhB transcripts were observed in the wild-type and mutant plants under normal growth conditions. The ratios of spliced and unspliced transcripts of ndhA and ndhB were much lower in rh3-4 and amiR1 mutant plants than in the wild-type plants under cold or salt stress compared with those under normal growth conditions (Fig. 5C), suggesting that splicing of ndhA and ndhB genes was much severely impaired in the mutants than in the wild-type plants under cold or salt stress conditions, (Fig. 5). By contrast, the ratios of spliced and unspliced transcripts of ndhA and ndhB in the wild-type and in rh34 and amiR1mutant plants under dehydration stress condition were similar to those under normal conditions (Supplemental Fig. S4), suggesting that splicing of ndhA and ndhB genes was not noticeably altered in the mutants compared with that in the wildtype plants under dehydration stress conditions. These results demonstrate that AtRH3 affects splicing of ndhA and ndhB introns specifically under cold or salt stress conditions. 3.5. AtRH3 possesses RNA chaperone activity Considering that RNA helicases can unwind double-stranded RNAs and have a potential to function as RNA chaperones in cells, we next determined whether AtRH3 possesses RNA chaperone activity. Several in vivo and in vitro assays are generally used to test the RNA chaperone activity of a protein (Semrad, 2011; Kang et al.,

2013). Here, we employed two in vivo and one in vitro assay to evaluate AtRH3 RNA chaperone activity. First, the complementation ability of AtRH3 in the RNA chaperone-deficient E. coli BX04 mutant cells was assessed. The colony-forming ability of E. coli BX04 mutant that lacks four cold shock proteins (CSPs), which function as RNA chaperones during cold adaptation (Bae et al., 2000; Phadtare et al., 2002) and is highly sensitive to cold temperatures (Xia et al., 2001), was evaluated with AtRH3 expression. When the BX04 cells were grown at normal growth temperature (37  C), the cells harboring each construct grew well with no noticeable differences. However, when the cells were incubated at low temperature (20  C), the BX04 cells expressing AtRH3 as well as E. coli CspA as a positive control grew well, whereas the BX04 cells harboring the pINIII vector as a negative control did not grow well at low temperature (Fig. 6A). This complementation ability of AtRH3 in coldsensitive BX04 cells suggests that AtRH3 has RNA chaperone activity. Second, a transcription anti-termination assay was used to further confirm AtRH3 RNA chaperone activity. E. coli strain RL211, which contains a chloramphenicol resistance gene downstream of the trpL terminator (Landick et al., 1990) and serves as an efficient system to test the transcription anti-termination activity of a protein (Bae et al., 2000; Phadtare et al., 2002). The RL211 cells harboring the pINIII vector alone did not grow on chloramphenicol medium, whereas RL211 cells expressing AtRH3 or CspA as a positive control grew on chloramphenicol medium (Fig. 6B). These results demonstrate that AtRH3 is capable of melting the RNA secondary structures present in trpL terminator, which confirms that AtRH3 has RNA chaperone activity. To further confirm the RNA chaperone activity of AtRH3, we finally measured the nucleic acidmelting ability of AtRH3. The recombinant glutathione S-transferase (GST)-AtRH3 fusion proteins were expressed and purified from E. coli (Supplemental Fig. S5) and reacted with partially double-stranded, fluorescence-labeled DNA molecules, which are designed to produce fluorescence signals upon DNA melting. The addition of recombinant GST-AtRH3 or GST-CspA proteins resulted in greatly increased fluorescence, whereas adding GST alone produced no fluorescence (Fig. 6C), suggesting that AtRH3 has the ability to disrupt base pairs in DNA. All of these results clearly demonstrate that AtRH3 harbors RNA chaperone activity. 4. Discussion Our current analyses of amiRNA knockdown mutants support previous findings utilizing the T-DNA insertion rh3-4 knockout mutant that AtRH3 is essential for chloroplast function and normal growth of Arabidopsis. Asakura et al. (2012) showed that reduced AtRH3 function causes a pale-green seedling phenotype, whereas complete loss leads to embryo lethality. A recent report by Lee et al. (2013) demonstrated that AtRH3 is involved in chloroplast ribosome biogenesis and ABA and salt stress responses. Our present data analyzing amiRNA knockdown mutants also clearly demonstrate that AtRH3 plays important roles in Arabidopsis seedling growth and response to cold and salt stresses via the regulation of the splicing of intron-containing transcripts in chloroplasts. A previous report showed that AtRH3 is involved in the splicing of several RNAs in chloroplasts, including trnI, trnA, rps12, and rpl21 (Asakura et al., 2012). Our present analysis showed that AtRH3 is involved in the splicing of not only trnI, trnA, rps12, and rpl21 but also atpF, trnL, and trnK (Fig. 4). The rps and rpl genes encode the small and large subunits of ribosomal proteins, and splicing deficiency of rps and rpl genes affects ribosome biogenesis and assembly (Schmitz-Linneweber et al., 2006; Asakura et al., 2012; Chi et al., 2012). The atpF is a subunit of proton-transporting ATP synthase complex that catalyzes the final step of oxidative phosphorylation and photophosphorylation (Gajadeera and Weber,

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Fig. 4. Abnormal splicing of chloroplast introns in rh3-4 mutant plants in normal growth conditions. (A) The splicing patterns of chloroplast intron-containing genes were analyzed by RT-PCR in the wild type (Col-0), rh3-4 mutant, and amiRNA transgenic lines (amiR1-1 and amiR1-2) under normal growth conditions. Exons and introns for each gene are represented as gray boxes and lines, respectively. Identical results were obtained from three independent experiments, and one representative result is shown. Amplification of tubulin transcripts in the same RNA samples is shown as a control. (B) The splicing patterns of atpF, trnL, and trnK were further analyzed by Northern blot analysis in the wild type, rh3-4 mutant, and amiRNA transgenic lines.(C) Splicing efficiency of atpF, trnL, and trnK transcripts in the wild-type and mutants plants was evaluated by real-time RT-PCR analysis using the primers that specifically amplify pre-RNAs and mature RNAs. The histogram shows the ratios of mature RNAs (spliced) and pre-RNAs (unspliced) in the mutant plants compared with those of wild-type plants. Data are mean ± SE obtained from three independent experiments.

2013). It is likely that decreased levels of rps12, rpl21, and atpF transcripts caused by splicing deficiency in AtRH3 mutants affect ribosome biogenesis and ATP synthesis, which results in retarded growth of the mutant plants under normal and stress conditions. Interestingly, splicing of ndhA and ndhB genes was severely impaired in rh3-4 and amiR mutants under cold and salt stress but not under dehydration stress conditions (Fig. 5 and Supplemental Fig. 4). Because seedling growth of rh3-4 and amiR mutants was much severely inhibited under cold and salt stress conditions than under dehydration stress conditions (Figs. 2 and 3), these results

suggest that defects in the splicing of ndhA and ndhB genes are related, at least in part, to the cold- and salt-sensitive phenotypes of the plant. Notably, the expression of ndhA and ndhB decreased under cold stress (Fig. 5B), which is in line with the observation that cold stress decreases the transcript levels of ndh gene in potato leaves (Svensson et al., 2002). The role in cold stress tolerance of chloroplast NAD(P)H dehydrogenase (NDH) (Li et al., 2004) and a nucleo-cytosolic DEAD-box RNA helicase (Guan et al., 2013) has been demonstrated. Mass spectrometry analysis revealed that chloroplast ndhA and ndhB interact with multiple copies of PSI to

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Fig. 5. Abnormal splicing of chloroplast introns in rh3-4 mutant plants under stress conditions. (A) The splicing patterns of ndhA and ndhB were analyzed by RT-PCR in the wild type (Col-0), rh3-4 mutant, and amiRNA transgenic lines (amiR1-1 and amiR1-2) under normal, cold (10  C), and salt (100 mM NaCl) conditions. Identical results were obtained from three independent experiments, and one representative result is shown. Amplification of tubulin transcripts in the same RNA samples is shown as a control. (B) The splicing patterns of ndhA and ndhB were further analyzed by Northern blot analysis in the wild type, rh3-4 mutant, and amiRNA transgenic lines under normal, cold (10  C), and salt (100 mM NaCl) conditions. (C) Splicing efficiency of ndhA and ndhB transcripts in the wild-type and mutants plants was evaluated by real-time RT-PCR analysis using the primers that specifically amplify pre-RNAs and mature RNAs. The histogram shows the ratios of mature RNAs (spliced) and pre-RNAs (unspliced) in the mutant plants compared with those of wild-type plants. Data are mean ± SE obtained from three independent experiments.

form the unique NDH-PSI supercomplex (Peng et al., 2011). It has been demonstrated by chloroplast ndhB mutant analysis that NDH complex functions for the protection of photosynthetic apparatus from photodamage (Endo et al., 1999). A recent report demonstrated that splicing of ndhB transcripts is strongly affected in the albino mutants (Tseng et al., 2013). It is becoming clear that RHs are important players in the splicing of introns in plant organelles. A phylogenetic analysis showed that AtRH3 is part of a clade with two closely related mitochondrial RH proteins, AtRH9 and AtRH53

(Matthes et al., 2007). AtRH53 has been demonstrated to function € hler et al., 2010). These in intron splicing of mitochondrial genes (Ko results clearly indicate that AtRH3 is an indispensible component of chloroplast splice factors, and suggest that proper splicing of chloroplast introns is important for plant adaptation to stresses. Mass spectroscopic identification of plastid-targeted proteins has revealed that seven Arabidopsis DEAD-box RHs, including AtRH3, AtRH22, AtRH26, AtRH39, AtRH47, AtRH50, and AtRH58, are localized to chloroplasts (Peltier et al., 2006; Zybailov et al., 2008;

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Fig. 6. RNA chaperone activity of AtRH3. (A) The colony-forming abilities of E. coli BX04 mutant cells harboring the pINIII vector (negative control), CspA (positive control), or AtRH3 were investigated at low temperature (20  C). The pictures were taken after 1day at 37  C and 6 days at 20  C. (B) Transcription anti-termination activity was measured in E. coli RL211 cells harboring each construct on LB agar plates with (þ) or without () chloramphenicol (Cm) at 37  C. The pictures were taken after 1 day for þ Cm and 4 days for e Cm. (C) DNA-melting ability of the proteins was assessed by measuring the fluorescence of a molecular beacon with the addition of GST (50 pmol), GST-CspA (50 pmol), or GST-AtRH3 (3 pmol).

Olinares et al., 2010). A sequence analysis of the seven RHs indicated that AtRH3 is closely related with mitochondrial RH9/RH53 and is in a clade distinct from the clade with five RHs (AtRH22, AtRH39, AtRH47, AtRH50, and AtRH58) and that AtRH26 is in a clade with proteins with unknown functions (Asakura et al., 2012; Chi et al., 2012). Analysis of the primary structures of RHs showed that AtRH3 and AtRH26 harbor all conserved motifs found in DEADbox RNA helicases, whereas the five AtRH proteins lack several conserved DEAD-box helicase motifs (Asakura et al., 2012). Although both AtRH3 and AtRH22 play similar roles in the biogenesis and assembly of the 50S ribosome (Asakura et al., 2012; Chi et al., 2012), their roles in RNA metabolism should be quite different. Mutation of AtRH22 results in no obvious differences in the levels of chloroplast RNA transcripts, including psbA (encoding D1), psbB (encoding CP47), psbD (encoding D2), atpB (encoding CF1b), psaA (encoding PsaA), and petA (encoding cytochrome f) (Chi et al., 2012). By contrast, splicing of several intron-containing chloroplast genes was impaired in AtRH3 mutants (Fig. 4 and Asakura et al., 2012). These results suggest that AtRH3 and AtRH22 exert their roles differently during chloroplast RNA metabolism. The most important unanswered question in research of chloroplast-localized RHs is how RNA helicases are involved in intron splicing of diverse chloroplast genes and, thus, affect chloroplast function and plant growth under normal and stress conditions. Our present data strongly suggest that AtRH3 exerts its role by

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functioning as an RNA chaperone. In cells, the functions of most RNAs rely on well-defined three-dimensional structures, and the correct splicing of introns requires the formation of splicingcompetent structures for substrate RNAs. However, correct folding of RNA molecules is an extremely complicated process that relies on diverse proteins assisting RNAs to reach their functionally active states. Several RNA-binding proteins, such as RNA chaperones, RNA annealers, and specific RNA-binding proteins have been implicated to play roles in the RNA folding process (Rajkowitsch et al., 2007). RNA chaperones are nonspecific RNA-binding proteins that bind diverse RNA substrates with low sequence specificity and can aid RNA folding by preventing RNA misfolding or by resolving misfolded RNA species (Semrad, 2011; Jung et al., 2013; Kang et al., 2013). The functional roles of RNA chaperones during intron splicing and development of plants have recently been demonstrated in Arabidopsis and rice (Kim et al., 2010; Kwak et al., 2012). DEAD-box RHs function as RNA chaperones that facilitate native folding of structured RNAs by accelerating conformational rearrangements (Linder and Jankowsky, 2011). In particular, several DEAD-box proteins in bacteria, yeast, and animals, including Mss116p, Ded1p, CYT-19, and SrmB, function as RNA chaperones that stimulates splicing of diverse group I and group II introns in mitochondria (Mohr et al., 2002; Bhaskaran and Russell, 2007; Halls et al., 2007; del Campo et al., 2009). The verified RNA chaperone activity of AtRH3 (Fig. 6) and the influence of AtRH3 on the splicing of a subset of chloroplast introns (Fig. 4 and Asakura et al., 2012) strongly suggest that AtRH3 plays a role as an RNA chaperone during chloroplast intron splicing. It is likely that, as an RNA chaperone, AtRH3 may assist in proper folding of intron-containing chloroplast genes to form the catalytically competent structures and avoid the formation of competing inactive structures, which is crucial for chloroplast function and the growth and stress response of plants. In conclusion, our results clearly demonstrate that AtRH3 plays important roles in chloroplast intron splicing and the growth and stress response in Arabidopsis. Considering that the functions of only limited Arabidopsis chloroplast-localized DEAD-box RHs have been demonstrated so far, future experiments should focus on determining the biological functions and cellular roles of other chloroplast-localized RHs in plants. In particular, it would be of interest to determine whether DEAD-box RHs play a role as an RNA chaperone during splicing and to uncover how these RHs are linked together to form protein complexes for correct splicing of chloroplast intron-containing genes. Acknowledgments We thank Drs. M. Inouye and S. Phadtare for the BX04 mutant cells and pINIII vector and Dr. R. Landick for the E. coli RL211 cells. This study was supported by grants from the Mid-career Researcher Program through a National Research Foundation of Korea grant funded by the Ministry of Education, Science and Technology (20110017357) and from the Next-Generation BioGreen21 Program (PJ00949102), Rural Development Administration, Republic of Korea. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2014.07.006. Authors' contributions H. Kang supervised the research design and wrote the paper; L. Gu, T. Xu, and K. Lee performed all molecular biology and biochemical experiments; K.H. Lee performed EM analysis.

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