# Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress
## 4 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  
Figure 6: Mutation of _Trx-h2_ in Arabidopsis disrupts mRNA export from the nucleus to the cytoplasm. The distribution of mRNAs was examined in the leaves of 2-week-old WT (Col-0), _trx-h2_, and Trx-h2 Oe Arabidopsis plants at 22 \({}^{\circ}\)C or 0 \({}^{\circ}\)C by in situ poly(A) mRNA hybridization. After fixation the leaves, 5\({}^{\prime}\)-fluorescein-labeled oligo(dT) probe was hybridized and the fluorescence of the leaf cells was observed using confocal microscopy (Olympus FV500) at excitation and emission wavelengths of 488 and 522 nm, respectively. Scale bars = 20 \(\upmu\)m.  
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 WCSPI 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.