Arabidopsis Disulﬁde Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

: The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulﬁde reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression ( Trx-h2 OE ) lines showed signiﬁcantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efﬁciently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.


Introduction
Since plants are sessile organisms, they have evolved sophisticated defense mechanisms to cope with unfavorable environmental conditions. Environmental stresses trigger a variety of defense signaling pathways involved in the production of reactive oxygen species (ROS) [1,2]. Depending on the concentration, ROS function as a double-edged sword, playing beneficial or detrimental roles in living organisms [3]. At a normal level, ROS participate in a number of signaling cascades that regulate diverse physiological and metabolic processes in plants to ensure optimal growth and development. However, excessive ROS induce damage to intracellular macromolecules and result in ER stress and ultimately cell death [4,5]. Therefore, to prevent apoptosis, ER stress must be regulated through UPR, such as refolding and ERAD, and a homeostatic level of ROS need to be maintained to function as secondary messengers, such as hormones, cyclic nucleotides, and calcium ions. [6,7]. To balance the level of ROS derived from both exogenous sources (temperature, drought, UV light, and various environmental stresses) and endogenous sources (chloroplast, mitochondria, and peroxisome), plants produce diverse non-enzymatic and enzymatic antioxidant molecules, including ascorbic acid, glutathione, and tocopherol [8], and a number of antioxidant proteins, including many isoforms of peroxidases, catalases, glutaredoxins, peroxiredoxins, and thioredoxins (Trxs) [9][10][11][12][13].
Trxs are a group of low molecular weight redox proteins ubiquitously distributed in eukaryotic and prokaryotic cells, which perform highly important functions in cells [14]. In plants, Trxs control transcription factors, activate ribonucleotide reductase, enhance photosynthetic efficiency, and regulate enzyme activities [15]. The following six major groups of Trx proteins have been identified in higher plants: m, f, x, y, o, and h [16,17]. In Arabidopsis thaliana, the Trx-h family contains eight cytosolic and nuclear isotypes, and each of these proteins exhibits a unique expression pattern and performs a specific function in response to various external stresses [18]. Trx-h1 and Trx-h4 are involved in cell cycle control and proliferation [19]. Trx-h3 is involved in plant defense under oxidative stress [20,21]. Trx-h5 plays a critical role in the immune response, and participates in the structural and functional regulation of NPR1 [22,23]. However, the specific function of many Trx-h isoforms remains unclear.
Here, we investigated the biochemical and physiological properties of Trx-h2, which contains an additional 19 amino acids at its N-terminus [24]. Our results demonstrate that Trx-h2 functions as an RNA chaperone under cold stress and plays a critical role in enhancing the cold tolerance of plants

Plant Materials and Growth Conditions
Plants of Arabidopsis thaliana ecotype Columbia (Col-0; wild-type (WT)) were used for characterizing the physiological properties of Trx-hs. The T-DNA insertion knockout mutant, trx-h2 (SALK_079509), was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, OH, USA) and used for generating Trx-h2 overexpression (Trx-h2 OE ) lines. Arabidopsis Trx-h3 overexpression lines and trx-h3 knockout mutant lines used as controls have been described previously [21]. All plants were grown under controlled conditions (22 • C temperature, 70% relative humidity, 16 h light/8 h dark photoperiod, and 100-120 µmol m −2 s −1 photosynthetic flux).

Western Blot Analysis
Crude protein extract of Arabidopsis was prepared using extraction buffer (100 mM Tris-HCl (pH 7.5), 1 mM EDTA, 20% glycerol, and 1 mM PMSF). Arabidopsis proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Immunoblot analysis was carried out using anti-Trx-h2 antibody prepared in our laboratory, and the Trx-h2 protein was detected by chemiluminescence. The antibody specificity of anti-Trx-h2 was demonstrated by using the Col-0, Vector, Trx-h2 OE , and trx-h2 plants.

Cold-Shock Treatment
Full-length cDNAs of Trx-h2 and Trx-h3 were amplified using the gene-specific primers (Trx-h2: 5 -CATATGATGGGAGGAGCTTTATCAACT-3 and 5 -GGATCCTTATGCTCTGA GTTTGCTAACT-3 ; Trx-h3: 5 -CATATGCATATGATGGGAGGAGCTTTATCAACT-3 and 5 -TCAAGCAGCAGCAACAACTGTCTTCTAACT-3 ) and cloned them into the pINIII vector. Each plasmid was transformed into the cold-sensitive E. coli BX04 cells that are deficient of the four cold-shock protein (csp) genes (cspA, cspB, cspE, and cspG) encoding the RNA chaperone proteins. The bacterial cells spreaded in Luria-Bertani (LB)-agar containing ampicillin were incubated at 37 • C, overnight. A single bacterial colony was picked and cultured for 12 h in LB-liquid media. Liquid culture cells were subjected to serial dilution and 5 µL of the cells were spotted onto LB-agar medium. Then the plates were incubated either at 37 • C for 1 day (control treatment) or at 19 • C for 5 days (cold-shock treatment).

Expression and Purification of Trx-h2 and Trx-h3 Recombinant Proteins
Full-length cDNAs of Trx-h2 and Trx-h3 were amplified by PCR using gene sequencespecific primers (Trx-h2: 5 -GGATCCATGGGAGGAGCTTTATCAACT-3 and 5 -AAGCTTT TATGCTCTGAGTTTGCTAACTTT-3 ; Trx-h3: 5 -GGATCCATGGCCGCAGAAG-3 and 5 -TCAAGCAGCAGCAACAACTGTCTTAACTTT-3 ) and cloned into the pGEX expression vector. The resulting plasmids were transformed into E. coli BL21 (DE3) pLysS cells. A single bacterial colony transforming the plasmid construct was grown in LB liquid medium at 37 • C. After the culture reached an optical density of 0.5-0.6 at 600 nm, protein expression was induced by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and the culture was grown at 30 • C for 4 h. The cells were harvested by centrifugation and resuspended in a buffer containing 1.8 mM KH 2 PO 4 (pH 8.0), 140 mM NaCl, 2.7 mM KCl, and 10 mM Na 2 HPO 4 . Cells were then disrupted by sonication, and recombinant proteins were purified using GSH agarose beads. The Trx-h2 and Trx-h3 proteins were eluted by thrombin cleavage, dialyzed against 20 mM HEPES-NaOH (pH 8.0) buffer, and used for biochemical analyses.

Electrophoretic Mobility Shift Assay (EMSA)
The purified recombinant Trx-h2 and Trx-h3 proteins were mixed with 100 ng of M13 mp8 phage single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), luciferase (Luc) mRNA, and Arabidopsis total RNAs in 10X EMSA DNA binding buffer (200 mM HEPES [pH 7.5], 750 mM NaCl, 10 mM EDTA, 25% glycerol, 2% Triton X-100, and 20 mM DTT), and incubated on ice for 15 min. Samples were then subjected to EMSA at a constant voltage of 100 V for ssDNA and dsDNA, and 50 V for Luc mRNA and Arabidopsis total RNAs. Nucleic acids (DNA and RNA) were then detected by ethidium bromide staining.

Melting Assay of Nucleic Acids
Molecular beacon used as a substrate in this melting assay contains two complementary DNA oligonucleotides. Each end of the two oligo-strands was labeled with fluorophore (FITC) or black hole quencher (BHQ1), respectively. To anneal the molecular beacon, two DNA oligonucleotides labeled with FITC and BHQ1 were mixed at 1:20 ratio as optimized in the previous paper [25] and denatured them at 95 • C for 2 min. The denatured DNAs dissolved in 10 mM Tris-HCl (pH 7.5) buffer containing 1 mM MgCl2 were annealed by incubating them on ice for 20 min. To detect the melting activity of Trx-h2, base-paired molecular beacon was reacted with Trx-h2, Trx-h3, CspA, or glutathione S-transferase (GST) protein (10 µM each) at room temperature. Additionally, the fluorescence was measured on a Spectra Max Gemini XPS spectrofluorometer (Molecular Devices) using a 96-well plate at excitation and emission wavelengths of 555 and 575 nm, respectively.

Transcription Anti-Termination Assay
The pINIII constructs containing the Trx-h2 and Trx-h3 cDNAs were used to perform the transcription anti-termination assay. Each plasmid was transformed into E. coli RL211 cells, and the transformed cells were grown overnight at 37 • C in LB liquid medium containing ampicillin and kanamycin. Each colony was spotted onto LB-agar medium supplemented with or without chloramphenicol. After growing the cells in the media at 37 • C for 2 days, we analyzed the transcription anti-termination activity by examining the cell growth in the presence of chloramphenicol.

In Situ Hybridization Assay
Poly(A) mRNA in situ hybridization was performed as described previously [26]. Briefly, leaves of 2-week-old WT (Col-0), trx-h2, and Trx-h2 OE plants were harvested and fixed in a fixation buffer containing 120 mM NaCl, 7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 , 2.7 mM KCl, 0.1% Tween-20, 80 mM EGTA, 5% formaldehyde, 10% DMSO, and 50% heptane. The fixed leaf samples were dehydrated and postfixed in the fixation buffer at room temperature for 30 min. Then, leaf samples were rinsed twice with fixation buffer containing no formaldehyde and once with 1 mL Hyb Plus hybridization buffer. Subsequently, the samples were prehybridized with 1 mL hybridization buffer at 50 • C for 1 h, and then hybridized with 5 pmol of 5 -fluorescein-labeled 45-mer oligo(dT) probe in hybridization buffer at 50 • C for more than 8 h. After hybridization, the fluorescence of leaf samples was observed under a confocal microscope at excitation and emission wavelengths of 488 and 522 nm, respectively. Each experiment was repeated at least three times, with similar results.

Seedling Growth under Cold Stress
WT (Col-0), trx-h2, and trx-h3 knock-out mutant plants, and overexpression lines of Trx-h2 (Trx-h2 OE ) and Trx-h3 (Trx-h3 OE ) Arabidopsis were sown on Murashige and Skoog (MS) medium supplemented with 3% sucrose. What is more, the seeds transforming pCAMBIA1300 vector only were used as a control. The plates were incubated at 4 • C for 3 days in the dark prior to germination. To test the effect of cold stress on seedling growth, plates were placed vertically in an incubator maintained at 12 • C.

Statistical Analysis
Root length data are presented as mean ± standard error (SE). Statistical analysis of the difference in root length between cold and normal conditions was examined using Student's t-test (p < 0.05).

Role of Trx-h2 in Seedling Growth under Cold Stress
There are five representative Trx-h members in Arabidopsis distributed both in the cytoplasm and nucleus that have two conserved active-site cysteine (Cys) residues ( Figure 1). Comparison of the amino acid sequences of Trx-hs revealed that Thx-h2 contains an additional 19 amino acids at its N-terminus and a putative nuclear localization signal (NLS) at their C-terminus estimated by the NLS-mapper database [27]. In contrast to Trx-h2, the other members of Trx-hs do not contain NLS, except Trx-h8, analyzed by the same program. The Trx-h2 protein has been shown to perceive intracellular ROS and to transmit the signal to downstream target molecules [24]. To investigate the physiological function of Trx-h2 in plants, we generated homozygous trx-h2 mutant lines from the trx-h2 knockout line (SALK_079507; Col-0 background) obtained from the ABRC (Ohio State University) and overexpressed the Trx-h2 gene in the trx-h2 mutant background. Knockout mutation of the Trx-h2 gene was confirmed by genotyping, and overexpression of Trx-h2 was verified by western blotting using anti-Trx-h2 antibody ( Figure 2).  Next, we examined the stress resistance of trx-h2 and Trx-h2 OE plants by measuring the root length of these plants under cold-shock treatments. The trx-h2, Trx-h2 OE , and WT (Col-0) plants showed no difference in root length at normal temperature (22 • C) (Figure 3a). However, under cold conditions (12 • C), the root length of trx-h2 knockout mutant lines was shorter than that of WT (Col-0) plants, whereas roots of Trx-h2 OE lines were significantly longer than those of the WT (Col-0) (Figure 3b,c). These results strongly suggest that Trx-2 plays a critical role in plant cold tolerance at low temperature.

Trx-h2 Overexpression Complements the Cold-Sensitive Phenotype of the E. coli BX04 Mutant
Based on the results of the cold-shock treatment (Figure 3), we verified the ability of Trx-h2 to impart cold tolerance in the cold-sensitive E. coli BX04 mutant cells that lack four Csp genes (CspA, CspB, CspE, and CspG); proteins encoded by these genes are essential for bacterial growth at low temperature [28]. In this experiment, E. coli BX04 cells were transformed with the pINIII empty vector (control) or with pINIII vector carrying Trx-h2 or Trx-h3, and incubated at 37 • C or 19 • C; Trx-h3 served as a control because it contains no extra N-terminal amino acids, unlike Trx-h2. No difference was observed in the growth of BX04 cells expressing Trx-h2, Trx-h3, or pINIII empty vector at optimal growing conditions (37 • C) (Figure 4a). However, at low temperature (19 • C), the survival rate of E. coli expressing Trx-h2 was approximately 100-fold higher than that of E. coli mutants expressing the pINIII empty vector or Trx-h3 (Figure 4b). In addition, the survival of cells expressing the CspA gene used as a positive control was almost similar with Trx-h2 expressing cells at 19 • C. These results explicitly demonstrate that Trx-h2 complements the cold-sensitive phenotype of E. coli BX04 mutant cells at low temperatures.

Trx-h2 Binds to Nucleic Acids
Next, we investigated how Trx-h2 confers enhanced cold tolerance in plants and microorganisms at the molecular level. Many studies have reported that the nucleic-and cytoplasmic-RNAs can be denatured at low temperature by their non-specific pairing to form misfolded hairpin structures [26]. Furthermore, resistance to cold shock is typically contributed by RNA chaperones, which exhibit several common characteristics. First, most of the RNA chaperones bind to nucleic acids including ssDNA and dsDNA. Second, RNA chaperones exhibit an anti-terminating mRNA transcription activity via their nucleic acid melting ability [29,30]. Based on these data, we examined whether the Trx-h2 protein expressed in E. coli interacts with M13 mp8 phage ssDNA and dsDNA, Luc mRNA, and Arabidopsis total RNAs (as substrates) in vitro [26].
Varying amounts of purified recombinant Trx-h2 were reacted with individual substrates in the reaction mixture containing binding buffer, and EMSAs were carried out using 0.8% agarose gels. Increasing amounts of Trx-h2 induced successive retardation of nucleic acid movement in agarose gels, indicating the binding of dsDNA, ssDNA, and total RNAs by Trx-h2 (Figure 5a-d) Additionally, excess of Trx-h3 (100 µg/µL; control) did not induce a shift in the position of nucleic acids, suggesting that Trx-h2 specifically interacts with ssDNA, dsDNA, and total RNAs. The results indicate that Trx-h2 binds to DNA and RNA, which is one of the peculiar properties of RNA chaperones.

Trx-h2 Melts to Nucleic Acids
To test whether Trx-h2 can dissociate double-stranded nucleic acids, we used two partially complementary double-stranded oligonucleotides, which together comprised a molecular beacon. In the synthetic DNA substrate of the molecular beacon, the end of one strand was labeled with a fluorophore, while that of the other strand was conjugated with the fluorescent quencher ( Figure S1). Since the fluorophore and quencher labeled at the end of the molecular beacon form a stem-loop structure, fluorescence is significantly extinguished by the quencher when the ends of the two strands form a stem-loop structure. However, melting of the annealed hairpin-like beacon by RNA chaperone allows the emission of the fluorophore because it is spatially separated from the quencher. To determine the nucleic acid melting activity of Trx-h2, we incubated the Trx-h2 protein with the molecular beacon (substrate) and measured the fluorescence emitted by the fluorophore. We also reacted the molecular beacon with GST and CspA as negative and positive controls, respectively. Both Trx-h2 and CspA remarkably increased the fluorescence intensity of the molecular beacon (Figure 5e). By contrast, Trx-h3 and GST did not increase the fluorescence intensity of the beacon, suggesting that Trx-h2 exhibits a strong dsDNA melting activity in vitro.
Next, we examined the nucleic acid melting activity of Trx-h2 in vivo by using E. coli RL211 cells containing the chloramphenicol acetyltransferase (CAT) gene preceded by a ρindependent trpL terminator. At the trpL termination sequences, two structural features of the transcribed RNA chain were detected. The transcribed RNA chain contained the GC-rich self-complementary area with several intruding nucleotides, which permitted to form a hairpin-like stem-loop structure. Immediately after the stem-loop secondary structure, several U bases were followed in the transcribed RNA. Then the hairpin-like structure interacted with the RNA polymerase and led to its stalling at the transcribing site. In particular, since the base pairs between the A bases in the template DNA and the 3 -U residues of the nascent RNA molecule are exceedingly precarious, they prompted the liberation of the RNA chain from the transcription complex at trpL termination sites, without proceeding the CAT gene transcription. The result makes the RL211 cells sensitive to chloramphenicol. However, when the RNA chaperone melted the hairpin-like stemloop structure of the nascent RNA chain, it allows the continuous transcription of RNA polymerase for the CAT gene. Then the bacteria can express the CAT protein and make the cells tolerant to chloramphenicol ( Figure S2). For investigating the RNA-melting activity of Trx-h2, we cloned Trx-h2 into the pINIII vector and transformed the DNA construct into RL211 cells. Using the bacterial cells expressing the protein, we examined their survival in LB-agar media containing chloramphenicol, comparing with the cells expressing the pINIII vector, CspA, and Trx-h3 as controls. Whereas the RL211 cells transforming the pINIII vector or Trx-h3 construct were not able to grow on LB-agar medium containing chloramphenicol, the cells expressing Trx-h2 and CspA grew well under the same conditions (Figure 5f). The results strongly suggested that Trx-h2 melted the hairpin-like secondary structure of trpL terminator located at immediately upstream of the CAT gene, which permitted the expression of CAT protein. Then, the bacterial cells expressing the CAT protein were able to grow at the presence of chloramphenicol. Taken together with the DNA-binding and melting activities of Trx-h2, our in vitro and in vivo data clearly show that the redox protein, Trx-h2 in Arabidopsis, functions as an RNA chaperone under cold conditions.

Trx-h2 Exports Nuclear mRNA to the Cytoplasm under Cold Stress
Because Trx-h2 harbors a putative NLS at its C-terminal end ( Figure 1) and functions as an RNA chaperone (Figures 4 and 5), we investigated its role in the export of mRNAs from the nucleus to the cytoplasm at different temperatures. Leaves of 14-day-old WT (Col-0), trx-h2, and Trx-h2 OE Arabidopsis plants were incubated with a 45-mer oligo(dT) probe labeled with fluorescein at its end, and traced the localization of poly(A) mRNAs by in situ hybridization, as described previously [26].
Under normal conditions (22 • C), fluorescence signals were uniformly distributed throughout the nucleus and cytoplasm in WT (Col-0), trx-h2, and Trx-h2 OE cells, indicating that mRNAs were effectively transported from the nucleus to the cytoplasm ( Figure 6, upper panel). When incubated at 0 • C for 2 days, although WT (Col-0) and Trx-h2 OE leaves showed no difference in the distribution of fluorescence signals between the cytoplasm and nucleus, parts of strong fluorescence signals were remained at the nucleus in trx-h2 leaves, suggesting that the export system of mRNAs transport was critically disrupted in the trx-h2 mutant plant, resulting in the accumulation of mRNAs in the nucleus (Figure 6, lower panel). Together, our data suggest that Trx-h2 has dual functions acting as a disulfide reductase and as an RNA chaperone, whose function should be controlled upon the environmental temperature changes.

Discussion
The Trx-h isoforms comprise the largest group in the plant Trx family. Although the physiological functions of most of these proteins have been predicted based on their intra-cellular localization and interaction partners [20,31,32], evidence proving these predictions is lacking [33]. Thus, determination of the localization patterns and specific interaction partners of Trx-h proteins under different environmental conditions is critical for understanding their roles in plant cells [16,21,22]. In this study, we investigated the physiological function of Trx-h2, which contains a 19-amino-acid extension at its N-terminus. Based on its subcellular localization in cytosol, ER/Golgi, and mitochondria [34,35], we found that Trx-h2 acts as an RNA chaperone under cold stress. Our previous result showed that one of the cold-tolerant nature of Arabidopsis is facilitated by Trx-h2-mediated expression of cold-responsive genes through its reductase activity [36]. However, in this study, we also propose that another function of Trx-h2 acting as an RNA chaperone can also contribute to the enhancement of cold tolerance in plants. In particular, the RNA chaperone function was also detected from the Trx-h2(C/S) mutant protein, suggesting that redox state of Trx-h2 did not give any effect on its RNA chaperone function.
To translate the genetic information of RNAs to proteins, they must maintain their structure as single-stranded poly-anionic forms. However, under cold stress conditions, RNA structures are misfolded to form non-native and non-functional conformations. Our results suggest that Trx-h2 functions as an RNA chaperone under cold stress by binding to misfolded and over-stabilized inactive RNAs. Then, Trx-h2 melts the hairpin-shaped RNAs and facilitates their refolding to form an active conformation. The resulting RNAs in their native form can be translated to produce cold resistant proteins, which confer enhanced cold tolerance in plants, similar to the rice (Oryza sativa L.) proteins, OsGRP4 and OsGRP6 [29].
Most of the RNA chaperones contain one of the following common motifs: RNA recognition motif, cold-shock domain, glycine-rich region, or Cys-Cys-His-Cys (CCHC) zinc-finger motif [37]. Although Trx-h2, originally defined as a disulfide reductase also functions as an RNA chaperone (according to our data), it does not contain any conserved motif or domain characteristic of RNA chaperones, such as the human peroxiredoxin 1 (hPrx1) protein [38]. While RNA chaperones enhance plant cold tolerance, they are also reported to play significant roles in plant growth and development; for example, Arabidopsis proteins AtRZ-1a and AtGRP4 regulate seed germination and seedling growth under cold stress [39,40], and the chloroplast-localized DEAD-box RNA helicase, AtRH3, participates in multiple steps of chloroplast development [41]. RNA chaperones, including AtGRP4 and AtGRP7 in Arabidopsis, OsZR2 in rice, and WCSP1 in wheat, also enhance plant tolerance to multiple stresses [30]. Therefore, the physiological functions of Trx-h2 in relation to diverse abiotic stresses and phytohormone signaling pathways should be investigated further. This study reveals a novel function of Trx-h2 as an RNA chaperone under cold stress. Thus, our results highlight the potential for generating cold tolerant crops by overexpressing the Trx-h2 gene using biotechnological approaches.
Supplementary Materials: https://www.mdpi.com/article/10.3390/app11156865/s1, Figure S1: Schematic representation of the molecular beacon used to test the DNA-melting activity of RNA chaperones. Figure S2: Schematic representation of the E. coli RL211 cells expressing the Trx-h2 for the chloramphenicol resistance.

Conflicts of Interest:
The authors declare no conflict of interest.