# Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress
## 3 Results  
### 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-h3 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-h3 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 \({}^{\circ}\)C) (Figure 3a). However, under cold conditions (12 \({}^{\circ}\)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. Figure 1: Comparison of the N-terminal amino acid sequences of the five representative Arabidopsis Trx-h proteins. (**a**) alignment of the N-terminal amino acid sequences of Arabidopsis Trx-h proteins. Two conserved cysteine (Cys) residues in the CXXC motif positioned between amino acids 59 and 62 are indicated in a blue box. The extra 19 amino acids at the N-terminus of Trx-h2 are outlined in a red box. Numbers above the alignment indicate the amino acid positions. (**b**) amino acid sequence of Trx-h2. Active Cys residues in the Trx motif are indicated in red, and the bipartite nuclear localization signal (NLS), identified using the NLS-mapper database, is outlined in red box. The asterisk (*) indicates the stop codon. Figure 2: Identification of _trx-h2_ knockout mutant lines and transgenic _Trx-h2_ overexpression (_Trx-h2 OE_) lines by PCR. (**a**) schematic showing the location of the T-DNA insertion in the _trx-h2_ mutant (SALK_079507), and gel image showing the results of PCR-based genotyping. T-DNA was inserted in the third exon of the _Trx-h2_ gene. Primers (F, R, and LBa1) used for genotyping the plants are indicated in the schematic. The _trx-h2_ knockout mutant lines #1 and #2 were confirmed by PCR. (**b**) schematic showing the construct used to generate _Trx-h2 OE_ lines, and results of western blot analysis. The _Trx-h2_ gene was expressed under the control of the cauliflower mosaic virus _35S_ promoter (CaMV35S) and octopine synthase terminator (OCS). The Trx-h2 protein was detected by western blotting using anti-Trx-h2 antibody. Rubisco was used as a loading control. Figure 3: Trx-h2 increases the root growth of Arabidopsis seedlings under cold condition. **(a,b)** Comparison of the root growth of WT (Col-0), _trx-h2_ knockout mutant, and _Trx-h2_ overexpression (_Trx-h2_OE) plants at 22 \({}^{\circ}\)C **(a)** and 12 \({}^{\circ}\)C **(b)**. Plants expressing _Trx-h3_ or _pCAMBIA1300_ vector were used as controls. Arabidopsis seedlings were grown on Murashige and Skoog (MS) medium at different temperatures for 15 days. Plates were oriented vertically and photographed. **(c)** root length of various genotypes at different temperatures.