Plant Physiology and Biochemistry 73 (2013) 405e411

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Research article

Functional characterization of a plastid-specific ribosomal protein PSRP2 in Arabidopsis thaliana under abiotic stress conditions Tao Xu a, Kwanuk Lee a, Lili Gu a, Jeong-Il Kim b, Hunseung Kang a, * a Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, 300 Yongbong-dong, Buk-gu, Gwangju 500-757, Republic of Korea b Department of Biotechnology and Kumho Life Science Laboratory, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 August 2013 Accepted 22 October 2013 Available online 30 October 2013

Plastids possess a small set of proteins unique to plastid ribosome, named plastid-specific ribosomal proteins (PSRPs). Among the six PSRPs found in Arabidopsis thaliana, PSRP2 is unique in that it harbors two RNA-recognition motifs found in diverse RNA-binding proteins. A recent report demonstrated that PSRP2 is not essential for ribosome function and plant growth under standard greenhouse conditions. Here, we investigated the functional role of PSRP2 during Arabidopsis seed germination and seedling growth under different light environments and various stress conditions, including high salinity, dehydration, and low temperature. The transgenic Arabidopsis plants overexpressing PSRP2 showed delayed germination compared with that of the wild-type plants under salt, dehydration, or low temperature stress conditions. The T-DNA insertion psrp2 mutant displayed better seedling growth but PSRP2overexpressing transgenic plants showed poorer seedling growth than that of the wild-type plants under salt stress conditions. No noticeable differences in seedling growth were observed between the genotypes when grown under different light environments including dark, red, far-red, and blue light. Interestingly, the PSRP2 protein possessed RNA chaperone activity. Taken together, these results suggest that PSRP2 harboring RNA chaperone activity plays a role as a negative regulator in seed germination under all three abiotic stress conditions tested and in seedling growth of Arabidopsis under salt stress but not under cold or dehydration stress conditions. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Arabidopsis thaliana Abiotic stress Plastic-specific ribosomal protein Ribosome function RNA-binding protein

1. Introduction Chloroplast gene expression is mainly regulated at posttranscriptional level, including RNA processing, splicing, editing, decay, and translational control, during which a variety of nuclearencoded proteins are targeted to chloroplasts and play fundamental roles in the regulation of chloroplast gene expression [1e4]. As plastids are derived from cyanobacteria through endosymbiosis, translation in plastids occurs on prokaryotic-type 70S ribosome consisting of a large (50S) subunit and a small (30S) subunit. The 30S subunit of the plastid ribosome consists of 21 proteins with orthologs in Escherichia coli. Among them, 12 proteins are plastidencoded and the other 9 proteins are encoded by the nuclear genome [5]. The 50S subunit of plastid ribosome contains 31

Abbreviations: PSRP, plastid-specific ribosomal protein; RBP, RNA-binding protein; RRM, RNA-recognition motif. * Corresponding author. Tel.: þ82 62 530 2181; fax: þ82 62 530 2069. E-mail address: [email protected] (H. Kang). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.027

ribosomal proteins with orthologs in E. coli. Nine of these are encoded by the plastid genes, whereas 22 are encoded by nuclear genes [6]. In addition, plastid ribosomes also contain other proteins termed plastid-specific ribosomal proteins (PSRPs) that have no orthologs in E. coli [5,6]. It has been demonstrated in spinach (Spinacia olercea) proteomes that four PSRPs, including PSRP1, PSRP2, PSRP3 and PSRP4, are associated with the 30S ribosomal subunit and that two PSRPs, including PSRP5 and PSRP6, are associated with the 50S ribosomal subunit [5e7]. The genome of Arabidopsis thaliana contains all six PSRPs present in higher plants, including PSRP1 (At5g24490), PSRP2 (At3g52150), PSRP3 (At1g68590), PSRP4 (At2g38140), PSRP5 (At3g56910), and PSRP6 (At5g17870) (Table 1). The biological functions of PSRPs are just beginning to be uncovered. It has been shown that PSRP1 is not a ribosomal protein but rather a ribosome binding translation factor that acts as a functional homolog of the E. coli cold-shock protein pY [8,9]. Recently, it has been demonstrated that PSRP3, PSRP4, and PSRP5 are genuine ribosomal proteins that have structural and functional roles in the ribosome, whereas PSRP2 and PSRP6 are non-essential PSRPs that are not

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Table 1 Comparison of PSRP proteins found in the Arabidopsis genome. Name Gene ID

No. of Putative domain amino acid

Proposed functiona

Homologyb (%)

PSRP1 PSRP2 PSRP3 PSRP4 PSRP5 PSRP6

308 253 166 118 148 106

Non-ribosomal Not essential Ribosomal protein Ribosomal protein Ribosomal protein Not essential

4e13 5e15 4e15 4e16 7e16 4e16

At5g24490 At3g52150 At1g68590 At2g38140 At3g56910 At5g17870

RaiA, PY, YfiA RRM PSRP-3_Ycf65 No domain found No domain found No domain found

a

Proposed function is based on reports by Sharma et al. and Tiller et al. [9,10]. The homology between each PSRP was predicted based on the amino acid sequences of all six PSRPs. b

required for ribosome accumulation and translation under normal growth conditions [10]. Although these recent studies have proposed the fundamental roles of PSRPs in ribosome function and plant growth and development, the other possible roles that PSRPs can play under normal and stress conditions have not been fully investigated. Among the six PSRPs found in Arabidopsis, PSRP2 is unique in that it harbors two RNA-recognition motifs (RRMs) found in diverse RNA-binding proteins (RBPs). The Arabidopsis genome encodes more than 200 RBPs [11]. A variety of RBPs plays important roles in growth, development, and the stress response in plants [12,13]. Here, we investigated whether PSRP2 plays role in growth and the response of Arabidopsis to various environmental cues, including different light conditions, salt, drought or low temperature stress. As RRM-type RBPs often exhibit RNA chaperone activity, we also tested whether PSRP2 has RNA chaperone activity. We provide evidence showing that PSRP2 possesses RNA chaperone activity and plays a role as a negative regulator during seed germination and seedling growth of Arabidopsis under abiotic stress conditions.

and Genevestigator (https://www.genevestigator.com/gv/plant. jsp)). The expression of PSRP2 was markedly downregulated in Arabidopsis under salt or dehydration stress conditions, and its expression was noticeably downregulated by cold stress (Supplementary Fig. S1). 2.2. psrp2 mutant and PSRP2-overexpresing transgenic plants do not respond to different light environments Tiller et al. analyzed a T-DNA tagged mutant (Salk_140803) for PSRP2 and reported that PSRP2 is not essential for ribosome function and plant growth under standard greenhouse conditions [10]. To further characterize the functional roles PSRP2 in plant response to diverse environmental cues, PSRP2-overexpressing transgenic Arabidopsis plants (35S::PSRP2) and the T-DNA tagged mutant examined by Tiller et al. were analyzed in more detail [6]. Downregulation and overexpression of PSRP2 in the knockdown (KD) mutant and three representative transgenic lines (OX3, OX8, and OX10), respectively, were confirmed by RT-PCR analysis (Fig. 2). Because PSRP2 is a chloroplast-targeted ribosomal protein, we first

2. Results 2.1. Characterization and expression analysis of PSRP2 The amino acid sequence analysis revealed that the six PSRPs present in Arabidopsis share low levels of sequence homology (4e 16%) with each other (Table 1). The analysis of the presence of specific functional motifs or domains showed that PSRP1 contained RaiY, PY, or YfiA domains and that PSRP3 contained PSRP-3_Ycf65 domain. However, PSRP 4, 5, and 6 contained no proposed functional domains (Table 1). The PSRP2 was unique in that it harbored two RRMs, the canonical RNA-binding motif found in diverse RBPs (Fig. 1A and Table 1). To examine the development stage-dependent and tissue-specific expression patterns of PSRP2, the transcript levels of PSRP2 were analyzed by RT-PCR. The results showed that PSRP2 expression levels were similar at different developmental stages (Fig. 1B) and that PSRP2 was expressed at similar levels in different organs (Fig. 1C). In a recent analysis, Tiller et al. showed by transient expression that PSRP2-GFP fusion protein is localized to chloroplasts in tobacco protoplasts [10]. To further confirm the subcellular localization of PSRP2, we generated transgenic Arabidopsis expressing the PSRP2-GFP fusion protein under control of the CaMV 35S promoter, and the subcellular localization of the fusion protein was analyzed by confocal microscopy. The result showed that strong GFP signals were detected exclusively in chloroplasts, confirming that PSRP2 is localized to chloroplasts (Fig. 1D). Stress-responsive expression patterns of PSRP2 were analyzed using the data in two public expression databases (Arabidopsis eFP Browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi)

Fig. 1. Domain structure, expression patterns, and subcellular localization of PSRP2 in Arabidopsis. (A) Schematic presentation of the PSRP2 domain structure. Two RNArecognition motifs (RRM) are shown. (B) Growth stage-dependent and (C) tissuespecific expression patterns of PSRP2 were analyzed by RT-PCR. 1, 2-leaf stage; 2, 6leaf stage; 3, 8-leaf stage; 4, bolting stage; 5, 5-week-old; 6, flowering stage; WP, whole plant; R, root; B, flower bud; S, stem; F, flower; RL, rosette leaf. (D) PSRP2-GFP fusion proteins were expressed in Arabidopsis, and GFP signals were detected using a confocal microscope. Bar ¼ 50 mm.

T. Xu et al. / Plant Physiology and Biochemistry 73 (2013) 405e411

reasoned that PSRP2 may play a role in light responses. To test this hypothesis, growth of wild-type, psrp2 KD mutant, and 35S::PSRP2 plants was evaluated under red light, far-red light, blue light, or dark conditions. The results showed no significant differences in seedling growth of the plants under these light conditions (Fig. 3A). In addition, growth of wild-type, psrp2 mutant, and 35S::PSRP2 plants was similar after UV-B treatment (Fig. 3B). All of these results indicate that PSRP2 plays no role in the light response of Arabidopsis. 2.3. PSRP2 affects seed germination of Arabidopsis under salt, dehydration, or cold stress conditions To investigate whether PSRP2 affects seed germination under various abiotic stress conditions, the germination rates of wildtype, psrp2 mutant, and 35S::PSRP2 seeds were evaluated on MS medium containing different concentrations of NaCl or mannitol. The germination rates of wild-type, mutant, and 35S::PSRP2 seeds were similar to each other on normal MS medium (Supplementary Fig. S2). However, when the seeds were germinated in the presence of NaCl or mannitol, germination of 35S::PSRP2 seeds was delayed compared to that of wild-type and mutant seeds. Approximately 40% of the mutant seeds germinated on day 2, whereas less than 20% of the 35S::PSRP2 seeds germinated on day 2 under the 100 mM NaCl condition (Fig. 4). The germination rates of wild-type and 35S::PSRP2 seeds were approximately 53% and 34%, respectively, on MS medium supplemented with 300 mM mannitol (Fig. 4). When the seeds were germinated at low temperature (10  C), the germination rates of wild-type and 35S::PSRP2 seeds were approximately 50% and 24% on day 5, respectively (Fig. 4). Germination rates of psrp2 mutant seeds were slightly higher than those of wild-type seeds under these stress conditions (Fig. 4). These results demonstrate that PSRP2 negatively affects Arabidopsis seed germination under salt, dehydration, or low temperature stress conditions.

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2.5. PSRP2 has the ability to bind ssDNA and RNA in vitro Because PSRP2 harbors two RRMs found in many RBPs, we determined whether PSRP2 has the ability to bind RNA and/or DNA. The recombinant glutathione S-transferase (GST)-PSRP2 fusion protein, the GST-CspA fusion protein (positive control), and the GST protein (negative control) were purified in E. coli (Supplementary Fig. S7), and their bindings to DNA or RNA were examined. When the GST-PSRP2 fusion proteins were mixed with DNAs, it was evident that PSRP2 protein binds to single-stranded (ss)DNA but not to double-stranded (ds)DNA (Fig. 6A). CspA, used as a positive control, also bound to ssDNA but GST alone did not bind to ssDNA and dsDNA (Fig. 6A). We next analyzed the binding of PSRP2 to 164 nucleotide-long synthetic RNA that was transcribed from pET22b(þ) plasmid using T7 RNA polymerase. Since the synthetic RNA derived from pET-22b(þ) plasmid has no biological function, it is commonly utilized as an RNA substrate to test sequencenonspecific binding of RBPs [14,15]. When GST-PSRP2 proteins were mixed with this synthetic RNA, the shifted bands were observed in the mixture of GST-PSRP2 protein, whereas no shifted bands were observed in the mixture of GST protein (Fig. 6B). These results clearly show that PSRP2 is capable of binding to both ssDNA and RNA possibly with a sequence-nonspecific manner. 2.6. PSRP2 possesses RNA chaperone activity Because PSRP2 binds to RNA and ssDNA (Fig. 6) and contains two RRMs found in many RBPs harboring RNA chaperone activity, we determined whether PSRP2 has RNA chaperone activity. We first assessed the ability of PSRP2 to complement the cold-sensitive phenotypes of RNA-chaperone deficient E. coli BX04 cells at low temperatures. The BX04 cells harboring PSRP2, a CspA that functions as an RNA chaperone during cold adaptation [16], or pINIII vector (negative control) grew well with no noticeable differences at normal temperature (data not shown). However, when these cells were subjected to cold shock at 19  C, it was evident that the

2.4. PSRP2 has a negative impact on seedling growth under salt stress conditions With the observation that psrp2 mutant and 35S::PSRP2 seeds germinated differently under salt, dehydration, or cold stress conditions, we further assessed seedling growth under stress conditions. After the seeds were fully germinated on MS medium for 3 days, the seedlings were transferred to MS medium supplemented with NaCl or mannitol, or the MS plates were placed in a growth chamber maintained at 10  C. No significant differences in seedling growth were observed among wild-type, psrp2 mutant, and 35S::PSRP2 plants when grown under dehydration or cold stress conditions (Supplementary Figs. S3 and S4). By contrast, the mutant plants displayed much larger and greener leaves than wild-type plants when grown under salt stress conditions, whereas 35S::PSRP2 plants showed smaller and yellowgreen leaves (Supplementary Fig. S5). The root lengths of 35S::PSRP2 plants were slightly shorter than those of wild-type and mutant plants (Fig. 5A). Moreover, post-germination survival rates of the seedlings were substantially different among the genotypes; survival rates of wild-type, psrp2 mutant, and 35S::PSRP2 seedlings were approximately 39%, 63%, and 17e30%, respectively, 25 days after growth on MS medium supplemented with 150 mM NaCl (Fig. 5B). Seed germination and seedling growth of wild-type, psrp2 mutant, and 35S::PSRP2 plants were not significantly different on MS medium supplemented with abscisic acid (Supplementary Fig. S6). These results demonstrate that PSRP2 negatively impacts Arabidopsis seedling growth under salt stress conditions.

Fig. 2. Confirmation of T-DNA tagged mutant and overexpression plants. (A) Schematic presentation of T-DNA mutant showing insertion of T-DNA (triangle) in the 50 UTR of PSRP2. Weak faint band was detected by RT-PCR, indicating the knockdown (KD) mutant. (B) Transcript levels of PSRP2 in Arabidopsis plants overexpressing PSRP2 (OX3, OX8, and OX10) were analyzed by RT-PCR.

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DNA base pairs. These results demonstrate that PSRP2 possesses RNA chaperone activity. 3. Discussion We demonstrated that PSRP2 harboring RNA chaperone activity affects seed germination and seedling growth of Arabidopsis under abiotic stress conditions. Downregulation of PSRP2 in Arabidopsis under salt, dehydration, or cold stress conditions (Supplementary Fig. S1) and retarded seed germination and seedling growth of PSRP2-overexpressing transgenic plants under stress conditions (Figs. 4 and 5) indicate that PSRP2 affects negatively seed germination and seedling growth of Arabidopsis under abiotic stress conditions. Tiller et al. analyzed a T-DNA tagged mutant and RNAi line to study the functional roles of PSRP2 [10]. The psrp2 mutants they analyzed maintained normal functions, including the maximum quantum efficiency of photosystem II (FV/FM), accumulation of thylakoid protein complexes, polysome loading, accumulation of ribosomal RNAs, and plastid ribosomal RNA accumulation and rRNA processing, which suggest that PSRP2 is a non-essential PSRP and is not required for ribosome accumulation and translation under normal growth conditions. However, the T-

Germination (%)

cells expressing PSRP2 or CspA grew well, whereas the BX04 cells harboring the pINIII vector did not grow well at low temperature (Fig. 7A). To further confirm PSRP2 RNA chaperone activity, we next evaluated its transcription anti-termination activity in the E. coli RL211 strain [17], which harbors a chloramphenicol resistance gene downstream from a trpL terminator and serves as an efficient system to test the RNA-melting activity of putative RNA chaperones [14,18,19]. The results showed that the RL211 cells expressing PSRP2 or CspA grew well on LB medium containing chloramphenicol, whereas the RL211 cells harboring pINIII vector did not grow on LB medium supplemented with chloramphenicol (Fig. 7B), indicating that PSRP2 and CspA can disrupt base pairs in RNAs. To further confirm PSRP2 RNA chaperone activity, the ability of PSRP2 to destabilize base pairs in partially double-stranded DNA substrates was assessed. The recombinant GST-PSRP2 fusion protein, the GSTCspA fusion protein, and the GST protein were purified (Supplementary Fig. S7), and used for the DNA-melting assay. Strong fluorescent signals were detected upon addition of the recombinant GST-PSRP2 or GST-CspA proteins to the reaction mixture. By contrast, the fluorescence signal did not increase upon addition of the GST protein (Fig. 7C), indicating that PSRP2 can melt

120 100 80 60 40 20 0

100 mM NaCl

Col-0 KD OX3 OX8 OX10

0

1

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6

Germination (%)

Days

120 100 80 60 40 20 0

300 mM mannitol

Col-0 KD OX3 OX8 OX10

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6

%) Germination (%

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120 100 80 60 40 20 0

Col-0 KD OX3 OX8 OX10

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Cold (10oC)

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6

7

Days Fig. 3. Response of the mutant and transgenic plants to light. (A) The wild type (Col-0), mutant (KD), and overexpression plants (OX3, OX8, and OX10) were grown under different light conditions, and the photograph was taken 7 days after the light treatment. (B) Six-day-old seedlings grown under normal conditions were grown further under UV light for 2 days, and the photograph was taken 14 days after recovery under normal conditions.

Fig. 4. Effect of various abiotic stresses on seed germination of mutant and transgenic plants. Seeds of wild type (Col-0), mutant (KD), and overexpression plants (OX3, OX8, and OX10) were germinated on MS medium supplemented with 100 mM NaCl or 300 mM mannitol at 25  C or on MS medium at 10  C, and germination rates (%) were scored on the indicated days. Values are means  SE of six replicates, approximately 100 seeds per replicate.

T. Xu et al. / Plant Physiology and Biochemistry 73 (2013) 405e411

DNA tagged knockdown mutant and RNAi line they analyzed were not complete loss-of-function mutants, and approximately 5e10% of PSRP2 was still expressed in the mutant lines [10]. It is possible that the small amount of PSRP2 expressed in the mutants was sufficient to display its proposed functions in plants. The PSRP2overexpressing transgenic plants analyzed in this study also did not show any noticeable phenotypes under normal growth conditions, but seed germination and seedling growth of the transgenic plants were retarded compared with those of wild-type plants (Figs. 4 and 5). Notably, PSRP2 affected seed germination under all stress conditions tested (salt, dehydration, or low temperature stress) but impacted seedling growth only under salt stress conditions, suggesting its different role as a negative regulator during seed germination or seedling growth of Arabidopsis under specific stress conditions. The importance of ribosomal proteins in plant growth under certain stress conditions has also been demonstrated in other cases. Ribosomal protein of the large subunit number 33, a plastid genome-encoded ribosomal protein, had no effect on plant viability and growth under standard conditions but was required for sustaining sufficient plastid translation capacity in the cold [20]. Chloroplast translation is complex and requires translation factors that do not have counterparts in bacteria. Proteomic and cryo-electron microscopy (EM) analyses of the chloroplast ribosome revealed that many chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs [21]. The location of PSRPs and their interaction with ribosomal components have been proposed by a cryo-EM study of the spinach chloroplast ribosome [8]. PSRP2 and PSRP3 are structural components of the 30S subunit, and PSRP2 and PSRP3 interact with each other and with 16S rRNA helices 6 and 10. PSRP4 occupies a position buried

Fig. 5. Effect of salt stress on seeding growth of the mutant and transgenic plants. (A) Root lengths of wild type (Col-0), mutant (KD), and overexpression plants (OX3, OX8, and OX10) were measured 5 days after growth on MS medium supplemented with 100 mM NaCl. Values are means  SD of at least 30 seedlings. (B) Seeds of wild type, mutant, and overexpression plants were sown on MS medium supplemented with 150 mM NaCl, and the survival rates (%) of seedlings were scored 25 days after germination. Values are means  SE of six replicates, approximately 40 seedlings per replicate.

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within the head of the 30S subunit. The 50S subunit possesses two small PSRPs, PSRP5 and PSRP6; PSRP5 is located near the tRNA-Exit site in a groove formed between 23S rRNA helices H68 and H88, and PSRP6 is relatively loosely associated with ribosomes. PSRP1 binds in the decoding region of the 30S ribosomal subunit, suggesting that PSRP1 is a translation factor rather than a ribosomal protein [8]. The physiological significance of PSRP2-16S rRNA interactions and the role of PSRP2 on 30S ribosomal subunit in chloroplasts are currently unclear. PSRP2 is the only PSRP that harbors RNA-binding domain (RBD) also called the RRM. The RRM contains a conserved ribonucleoprotein (RNP) 1 and RNP2 [22], which are important elements for recognizing target RNAs. PSRP2 shares a high sequence similarity with several RBPs, including chloroplast stroma RNA-binding proteins, polyadenylate-binding proteins, glycine-rich RNA-binding proteins, heterogeneous nuclear RNPs, and small nuclear RNPs [7]. RBD-containing proteins are generally associated with RNA metabolism, including processing, splicing, editing, transport, and turnover. In the U1A protein, the aromatic residues of RNP1 and RNP2 are critical for the interaction with base-stacked RNA substrates [22]. As the aromatic residues in the RRMs of PSRP2 are conserved, it was proposed that PSRP2 performs similar RNAbinding activity, and a cryo-EM study of spinach chloroplast ribosomes suggested that PSRP2 interacts with 16S rRNA [8]. Our results clearly show that PSRP2 is capable of binding to RNA and ssDNA (Fig. 6). Notably, PSRP2 possesses RNA chaperone activity (Fig. 7). RNA chaperones are nonspecific RBPs that interact with diverse RNA substrates with low sequence specificity and play a role in RNA folding process via structural rearrangement of target RNAs [23e25]. The binding capability of PSRP2 to the synthetic RNA derived from pET vector as well as to ssDNA (Fig. 6) strongly suggests that PSRP2 binds to RNAs with low sequence specificity. It is likely that the RNA chaperone activity of PSRP2 is needed for the

Fig. 6. Nucleic acid-binding ability of PSRP2. (A) Single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) was incubated with the recombinant GST-PSRP2, GST (negative control), or GST-CspA (positive control) proteins, and the binding complexes were separated on 1% agarose gel. The DNAs were visualized under UV. (B) The 33Plabeled synthetic RNAs were incubated with the recombinant GST-PSRP2 or GST (negative control) proteins, and the binding complexes were separated on 10% polyacrylamide gel. The RNAs were detected by Phosphorimager.

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regulation of RNA metabolism of diverse chloroplast RNA transcripts, which affects seed germination and seedling growth of plants under stress conditions. In conclusion, our results demonstrate that PSRP2 plays a role in seed germination and seedling growth of Arabidopsis under abiotic stress conditions. Although knockdown mutants and overexpression transgenic plants displayed only marginal phenotypes under normal and stress conditions, it is still possible that PSRPs are deeply involved in other cellular processes. Considering that PSRP2 is the only PSRP that harbors RRMs necessary for RNA binding and possesses RNA chaperone activity, it would be interesting to further investigate the functional roles of PSRP2 and to search for potential RNA targets and determine the mechanistic role of PSRP2 in the regulation of chloroplast RNA metabolism. 4. Materials and methods 4.1. Plant materials and growth conditions A. thaliana (Col-0 ecotype) was grown in a growth chamber at 23  C under long day conditions (16 h light/8 h dark). Seed germination and seedling growth under stress conditions were conducted essentially as described previously [14,26]. Sterilized seeds were sown on half-strength Murashige Skoog (MS) medium

supplemented with NaCl at 75e175 mM or with mannitol at 200e 300 mM, respectively, for the salt or dehydration stress treatments. For the low temperature stress treatment, seeds on MS medium were kept in a growth chamber maintained at 10  C. The seeds were regarded as germinated when the radicles protruded from the seed coat. To determine the effect of salt or dehydration stress on seedling growth, the seeds were fully germinated under normal conditions, and 4-day-old seedlings were transferred to MS medium supplemented with NaCl or mannitol. To determine the effect of cold stress on seedling growth, 4-day-old seedlings germinated under normal conditions were placed in a growth chamber maintained at 10  C. To determine the effect of different light wavelength on seedling growth, the seeds were fully germinated under normal conditions, and MS plates containing 10-day-old seedlings were kept in a growth chamber under different light environments, including red, far-red, blue, and dark conditions. Monochromic farred (lmax 738 nm), red (lmax 654 nm), and blue (lmax 470 nm) lights were provided from light emitting diode in a plant growth chamber (VS-9108M-LED, Vision Scientific Co. Seoul, Korea). The photon fluence rate of each light was 20 mmol m2 s1. 4.2. Vector construction and Arabidopsis transformation The full-length PSRP2 cDNA was cloned into BamHI/SalI site of the pCAMBIA 1301 vector, which overexpresses PSRP2 under the control of the CaMV 35S promoter. Arabidopsis transformation was performed according to the vacuum infiltration method [27] using Agrobacterium tumefaciens GV3101. To identify transgenic plants, seeds were harvested and plated on selection medium containing hygromycin (50 mg ml1) and carbenicillin (250 mg ml1). Overexpression of PSRP2 in the transgenic plants was confirmed by reverse transcription-PCR (RT-PCR) analysis, and T3 or T4 homozygous lines were used for phenotype investigation. 4.3. RNA extraction and RT-PCR Total RNA was extracted from the frozen plant samples using the Plant RNeasy Extraction kit (Qiagen, Valencia, CA, USA). RT-PCR was carried out using Qiagen One-step RT-PCR kit to detect PSRP2 transcripts at different development stages and in various tissues. The primers used for RT-PCR were as follows: for PSRP2, forward primer (50 -GCCGTCGATTTGGGTTTGC-30 ) and reverse primer (50 CGAATCCAAACCCGGTGG-30 ); for actin, forward primer (50 CTCCGTGTTGCTCCTGAGGAACATC-30 ) and reverse primer (50 ACCTCAGGACAACGGAATCGCTC-30 ). 4.4. RNA chaperone assay

Fig. 7. RNA chaperone activity of PSRP2. (A) Complementation ability of PSRP2 in the RNA chaperone-deficient E. coli BX04 mutant. The diluted cultures (101e105 dilution) of BX04 cells harboring PSRP2, CspA (positive control), or the pINIII vector (negative control) were spotted on LB-agar plates and incubated at 19  C. The photograph was taken 6 days after incubation. (B) Liquid cultures of RL211 cells harboring each construct were spotted on LB agar with (þ) or without () chloramphenicol (Cm), and the cells were grown at 37  C. The photograph was taken 3 days after incubation. (C) DNA-melting activity was determined by measuring the fluorescence (RFU, relative fluorescence unit) of a molecular beacon after adding GST-PSRP2, GST-CspA (positive control), or GST (negative control) proteins.

To construct an expression vector for the cold shock and transcription anti-termination assays in E. coli, the coding region of PSRP2 was cloned into the NdeI/BamHI site of the pINIII vector [28]. The cold shock and transcription anti-termination assays were conducted essentially as described previously [14]. For the cold shock assay, E. coli BX04 mutant cells [29], which lacked four cold shock proteins and are highly sensitive to cold stress, were transformed with each vector, grown in LB medium containing ampicillin and kanamycin until the optical density reached 1.0 at 600 nm, and the diluted cultures were spotted on LB-agar plates containing 0.4 mM IPTG and grown at 19  C. For the transcription anti-termination assay, E. coli RL211 cells transformed with each construct were grown at 37  C on LB-carbenicillin plates with or without chloramphenicol [17]. For the nucleic acid-melting assay, the molecular beacon with a fluorophore (tetramethyl rhodamine) and a quencher (dabcyl) was synthesized as described previously [14,18]. The recombinant GST-

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PSRP2 fusion proteins were expressed using the pGEX-4T-3 vector (Amersham Pharmacia Bio-sciences, Piscataway, NJ, USA) in BL21 DE3 cells, and were purified with glutathione Sepharose 4B resin. The fluorescence signals arising from the incubation of the molecular beacon with the GST-PSRP2 proteins were measured by a Spectra Max GeminiXS spectrofluorometer (Molecular Devices, San Diego, CA, USA) at excitation and emission wavelengths of 555 nm and 575 nm, respectively. All other experimental conditions were essentially as described previously [14]. 4.5. In vitro nucleic acid-binding assay The binding ability of PSRP2 to DNAs or RNAs was conducted as previously described [14,15]. For DNA binding assay, the recombinant GST-PSRP2 fusion proteins were mixed with either singlestranded (ss)DNA or double-stranded (ds)DNA in the binding buffer (10 mM TriseHCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 7% glycerol), the DNAeprotein complexes were separated on 1% agarose gel, and DNAs were visualized by UV. For RNA binding assay, the 33P-labeled RNA substrates were synthesized from pET22b(þ) vector using T7 RNA polymerase and were incubated with the recombinant GST-PSRP2 fusion proteins in the binding buffer. The RNAeprotein complexes were separated on 10% nondenaturing polyacrylamide gel, and RNA bands were detected using a Typhoon FLA7000 Phosphorimager (GE Healthcare Life Sci.). 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 the Mid-career Researcher Program through the National Research Foundation of Korea grant funded by the Ministry of Education, Science, and Technology (2011-0017357) and by a grant from the Next-Generation BioGreen 21 Program (PJ00820303), 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.2013.10.027. References [1] J. Marin-Navarro, A.L. Manuell, J. Wu, S.P. Mayfield, Chloroplast translation regulation, Photosynth. Res. 94 (2007) 359e374. [2] A.F. de Longevialle, I.D. Small, C. Lurin, Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles, Mol. Plant 3 (2010) 691e705. [3] D.B. Stern, M. Goldschmidt-Clermont, M.R. Hanson, Chloroplast RNA metabolism, Annu. Rev. Plant Biol. 61 (2010) 125e155. [4] A. Barkan, Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold, Plant Physiol. 155 (2011) 1520e1532. [5] K. Yamaguchi, K. von Knoblauch, A.R. Subramanian, The plastid ribosomal proteins: identification of all the proteins in the 30S subunit of an organelle ribosome (chloroplast), J. Biol. Chem. 275 (2000) 28455e28465. [6] K. Yamaguchi, A.R. Subramanian, The plastid ribosomal proteins: identification of all the proteins in the 50S subunit of an organelle ribosome (chloroplast), J. Biol. Chem. 275 (2000) 28466e28482.

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