Comparative Functional Analysis of Wheat (Triticum aestivum) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.

Introduction Gene expression at the posttranscriptional level, including RNA processing, splicing, transport, decay, and translation control, is regulated directly or indirectly by a variety of RNA-binding protein (RBPs) [1,2]. Typical RBPs contain one or two RNArecognition motifs (RRMs) at their N-terminus and various auxiliary motifs or regions at their C-terminus, such as glycinerich region, arginine-rich motif, RGG box, zinc finger motif, and SR-or RD-repeat regions [3]. According to recent progress in genome sequencing of plants, many RBPs have been identified in different plant species, including Arabidopsis thaliana, rice (Oryza sativa), wheat (Triticum aestivum), tobacco, rapeseed, and Camelina. It has been demonstrated that these RBPs are involved in genome organization, plant development, and stress responses [4][5][6].
Glycine-rich RNA-binding proteins (GRPs) are characterized by the presence of 1-3 RRMs at the N-terminus and a glycine-rich region at the C-terminus, which play a prominent role in plant responses to diverse abiotic and biotic stresses [4][5][6]. Among the different GRP family members, one GRP family, designated RZs, harbors a CCHC-type zinc finger motif between the N-terminal RRM domain and the C-terminal glycine-rich region [3,7]. The functional roles of RZs in plant stress response and adaptation have been demonstrated in Arabidopsis and rice [8][9][10][11]. Arabidopsis AtRZ-1a is strongly up-regulated by cold stress and down-regulated by dehydration stress or exogenous abscisic acid [8], and affects seed germination and seedling growth of plants under salt, dehydration, or cold stress [8,11,12]. In comparison, neither loss-of-function nor overexpression of AtRZ-1b or AtRZ-1c affects seed germination and seedling growth of plants under stress [9]. The rice genome encodes three RZ genes, named OsRZ1, OsRZ2, and OsRZ3. The transcript levels of all three OsRZs are up-regulated by cold stress, but are not significantly affected by drought or high salt stress [10]. OsRZ2, but not OsRZ1 and OsRZ3, possess RNA chaperone activity and rescue the coldsensitive Arabidopsis grp7 mutant from cold and freezing damage [10]. Although all of these previous reports clearly point to the important roles of RZ family members in the response of certain plant species to different stresses, the stress-responsive roles of RZs in other crop plants, including wheat, have yet to be determined.
The wheat genome harbors four genes encoding RZ proteins, named TaRZ1 (AF315811), TaRZ2 (AK335985), TaRZ3 (tplb0013k12), and TaRZ4 (AK331499). The TaRZ4 is unusual in that it encodes several different open reading frames with initiation and stop codons at different positions. Our previous study showed that TaRZ2, but not TaRZ3, has RNA chaperone activity [13]. In this study, the functional roles of three wheat RZ family members (TaRZ1, TaRZ2, and TaRZ3) were investigated using transgenic Arabidopsis plants that express each TaRZ gene under control of the cauliflower mosaic virus 35S promoter. Seed germination, seedling growth, and freeze tolerance of the transgenic plants were compared with each other under various stress conditions, and the functional roles of different TaRZ family members were comparatively analyzed in plants under abiotic stress conditions.

Isolation and characterization of TaRZs in wheat
The cDNA sequences of TaRZ1 (AF315811), TaRZ2 (AK335985), and TaTZ3 (tplb0013k12) encode a putative protein of 237, 211, and 293 amino acid residues with a predicted molecular mass of 25.8, 23.0, and 31.9 kDa, respectively ( Table 1). All three TaRZs contained well-conserved ribonucleoprotein1 (RNP1) and RNP2 sequences, as well as a C-terminal glycine-rich region, interspersed by a CCHC-type zinc finger ( Figure S1A). Sequence alignment showed that TaRZ1 and TaRZ2 have approximately 67% amino acid sequence homology, TaRZ1 and TaRZ3 have approximately 45% amino acid sequence homology, and TaRZ2 and TaRZ3 have approximately 43% amino acid sequence homology (Table 1). Amino acid sequence homology among the three TaRZs, which was calculated using a web-based program (http://www.uniprot.org/align), was approximately 25%. A phylogenetic analysis was conducted to examine the sequence similarity of RZ proteins among wheat, Arabidopsis, cabbage (Brassica rapa), rice, and maize (Zea mays). The results showed that wheat RZ sequences are more homologous to rice and maize RZ sequences than Arabidopsis and cabbage RZ sequences ( Figure S1B), suggesting that RZ proteins in monocot plants are highly conserved.
Transcript levels, stress-responsive expression patterns, and subcellular localization of TaRZs in wheat The transcript levels of three TaRZs in the aerial part of 2-weekold wheat seedlings were determined via quantitative real-time RT-PCR analysis. Among the three TaRZs in wheat, TaRZ2 showed the lowest level of expression under normal growth conditions, and the expression levels of TaRZ1 and TaRZ3 were approximately 64-and 14-fold higher than that of TaRZ2, respectively ( Figure 1A). The stress-responsive expression patterns of TaRZs were then assessed in wheat under salt, dehydration, or cold stress conditions. Wheat plants were subjected to the indicated stress treatments, and the expression levels of TaRZs were analyzed by real-time RT-PCR. To compensate for any circadian effect on gene expression, we measured the expression levels of TaRZs in non-stressed control plants as well as in stresstreated plants at each time point. Under salt stress conditions, the transcript levels of TaRZ1 and TaRZ2 decreased, whereas the transcript levels of TaRZ3 increased ( Figure 1B). Dehydration treatment marginally up-regulated TaRZ1 and TaRZ3 transcript levels ( Figure 1C). TaRZ expression was markedly up-regulated by cold stress as TaRZ1 and TaRZ2 transcript levels increased up to 7or 17-fold, respectively, 48 h after cold treatment, while transcript levels of TaRZ3 increased 2.5-fold by cold treatment ( Figure 1D).
To determine the subcellular localization of TaRZ proteins, cellular localization was first predicted via the PSORT (http:// psort.ims.u-tokyo.ac.jp) and TargetP (http://www.cbs.dtu.dk/ services/TargetP) programs. The results showed that TaRZ1  Table 1. Compilation of TaRZs investigated in this study. and TaRZ2 proteins were predicted to be localized to the nucleus, and TaRZ3 protein was predicted to be localized mainly to the nucleus and possibly to the chloroplast. To confirm the subcellular localization of TaRZ proteins, TaRZs-green fluorescent protein (GFP) fusion proteins were transiently expression in tobacco under control of the CaMV 35S promoter, and the localization of the fusion proteins was analyzed by confocal microscopy. The results showed that strong GFP signals were detected exclusively in the nucleus of TaRZ1-GFP and TaRZ3-GFP plants ( Figure 2). In comparison, GFP signals in TaRZ2-GFP plants were detected in several places, including the nucleus, endoplasmic reticulum (ER), and cytoplasm ( Figure 2). Localization of TaRZ2 in the ER was further supported by co-transformation of the tobacco leaves with microsomal delta-12 fatty acid desaturase (FAD2) that is localized to the ER and catalyzes the first committed step of the biosynthesis of polyunsaturated fatty acids [14] ( Figure 2). These results indicate that the TaRZ1 and TaRZ3 proteins are localized to the nucleus, whereas the TaRZ2 protein is localized to the nucleus, ER, and cytoplasm.

TaRZs affect seed germination of Arabidopsis under salt or dehydration stress conditions
With the observation that expression levels of TaRZs are regulated by different stress conditions, we examined whether TaRZs play any roles in plant stress responses. Because wheat transformation is technically difficult, we investigated TaRZ function using Arabidopsis. Multiple transgenic Arabidopsis plants expressing each TaRZ under control of the cauliflower mosaic virus 35S promoter were generated, and 3 T 3 homozygote transgenic lines were selected for phenotypic analysis. Expression of TaRZs in each transgenic line was confirmed by RT-PCR analysis ( Figure S2). Transgenic Arabidopsis plants expressing TaRZ1, TaRZ2, or TaRZ3 under control of the 35S promoter were designated 35S::TaRZ1, 35S::TaRZ2, or 35S::TaRZ3, respectively. To investigate whether TaRZ affects seed germination under various abiotic stress conditions, germination rates of the wild-type and each transgenic seed were evaluated on MS medium containing different concentrations of NaCl or mannitol. Germination rates of the wild-type and transgenic seeds were similar to each other on normal MS medium. However, when the seeds were germinated on MS medium containing NaCl, germination of all transgenic seeds was delayed compared with that of the wild-type seeds ( Figure 3). Dehydration stress affected germination of the transgenic seeds differently as germination of 35S::TaRZ2 and 35S::TaRZ3 seeds was delayed compared with that of the wild-type seeds, whereas germination of 35S::TaRZ1 seeds was similar to that of the wild-type seeds on MS medium supplemented with mannitol ( Figure 3). When the seeds were germinated at low temperature (10uC), germination rates of all transgenic seeds were similar to that of the wild-type seeds ( Figure S3). These results demonstrate that TaRZs affect seed germination differently under salt, dehydration, or cold stress conditions.

TaRZ1 and TaRZ2 have a negative impact on seedling growth under salt or cold stress conditions
As it is evident that TaRZs affected seed germination under stress conditions, we next assessed whether TaRZs play a role in seedling growth under stress conditions. Seeds were first fully germinated on MS medium for 3 days, and the seedlings were transferred to MS medium supplemented with NaCl or mannitol, or the MS plates were placed in a growth chamber maintained at 10uC for stress treatment. No significant differences in seedling and root growth were observed between the wild-type and 35S:: TaRZ3 plants under all stress conditions ( Figure S4). By contrast, seedling growth of 35S:: TaRZ1 plants was significantly inhibited under cold or salt stress conditions ( Figure 4A), and seedling growth of 35S:: TaRZ2 plants was also inhibited by salt stress ( Figure 4B). Dehydration stress did not affect seedling growth of 35S:: TaRZ1 plants ( Figure S5A), and dehydration or cold stress had no effect on seedling growth of 35S::TaRZ2 plants ( Figure S5B). These differences in seedling growth among the genotypes were consistently observed when tested on MS medium supplemented with different concentrations of NaCl or mannitol. These results demonstrate that TaRZ1 and TaRZ2 negatively affected seedling growth under salt stress conditions and that TaRZ1 negatively influenced seedling growth under cold stress conditions.

TaRZ2 confers freeze tolerance in Arabidopsis
Because expression of TaRZs was highly up-regulated in wheat under cold stress conditions, TaRZs are likely involved in the response of plants to cold or freezing stress. Although TaRZs did not affect seed germination and seedling growth of Arabidopsis under cold stress conditions ( Figures S3-S5), we next evaluated the functional role of TaRZs under freezing stress conditions. When the plants were subjected to freezing stress at 26 or 27uC for 4-24 h, survival rates and freeze tolerance of 35S::TaRZ1 and 35S::TaRZ3 plants were similar to those of the wild-type plants.
By contrast, survival rates and freeze tolerance of 35S::TaRZ2 plants increased compared with those of the wild-type plants. In non cold-acclimated (NA) freeze tolerance tests, approximately 40% of the wild-type plants survived, whereas 70-80% of 35S::TaRZ2 plants survived after freezing stress at 26uC for 4 h ( Figure 5A). In addition, in cold-acclimated (CA) freeze tolerance tests, approximately 55% of the wild-type plants survived, whereas 80-90% of 35S::TaRZ2 plants survived after freezing stress at 2 7uC for 16 h ( Figure 5B). Freeze tolerance of 35S::TaRZ2 plants was consistently observed when CA freezing tests were repeated at 27uC for 12-24 h ( Figure S6). To further confirm the role of TaRZ2 in freeze tolerance, the contribution of TaRZ2 to the enhanced freeze tolerance of Arabidopsis was evaluated by measuring cellular damage of the plants as a result of freezeinduced membrane lesions. Electrolyte leakage from the leaves of 35S::TaRZ2 plants was much less than that observed in the wildtype when incubated at 21 to 210uC ( Figure 5C). All of these results indicate that 35S::TaRZ2 plants are more tolerant to freezing stress than wild type, suggesting that TaRZ2 confers freeze tolerance in Arabidopsis.

Discussion
Our results show that different TaRZ family members play varying roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress conditions. Despite the potential role of RZ family members in various plants under stress, the experimental evidences supporting their functional roles are limited to only a few plant species, including Arabidopsis and rice. It has been reported that, among the three Arabidopsis AtRZs, AtRZ-1a plays a positive role in plant response to cold and freezing stress but negatively affects seed germination and seedling growth of Arabidopsis under salt or dehydration stress [8,11,12]. By contrast, AtRZ-1b or AtRZ-1c does not affect seed germination and seedling growth of Arabidopsis under stress conditions [9]. Here, we demonstrated that wheat TaRZ2 contributes to enhance freeze tolerance of Arabidopsis ( Figure 5) and plays a negative role during seed germination and seedling growth under salt or dehydration stress conditions (Figure 3 and 4). Sequence analysis shows that TaRZ2 is most homologous to AtRZ1-a among the three AtRZs ( Figure S1). Importantly, the cellular localization of TaRZ2 and AtRZ-1a was similar in that they were localized to the nucleus and cytoplasm, which is different from the nucleus localization of other Arabidopsis [9] and wheat TaRZs (Figure 2). These present and previous results suggest that AtRZ-1a and TaRZ2 are functionally conserved in Arabidopsis and wheat. Interestingly, the up-and down-regulation of TaRZs by cold, salt, or dehydration stress was similar to the stress-responsive expression patterns of AtRZs [8] and OsRZs [10]. We showed previously that, among the three rice OsRZs, only OsRZ2 has the ability to confer cold and freeze tolerance in Arabidopsis [10]. All of these results suggest that certain RZ family members are functionally conserved in diverse plant species during the cold and freeze stress response and adaptation. The next important question is to determine how RZs exert their roles during stress adaptation. Although we do not understand conclusively the mechanistic roles of RZs in the response of plants to all stresses, the function of RZs during cold adaptation could be related to the RNA chaperone activity of RZ proteins. RNA chaperones are non-specific RBPs that interact with diverse RNA substrates and aid RNA folding or structural rearrangement [15,16]. The function of RNA chaperones is particularly important during cold adaptation in E. coli, during which cold shock proteins are highly induced and function as RNA chaperones that destabilize over-stabilized secondary structures in mRNAs, thereby facilitating efficient translation at low temperature [17][18][19][20]. Notably, among the three TaRZ proteins in different plant species, only TaRZ2, OsRZ2, and AtRZ-1a in wheat, rice, and Arabidopsis, respectively, which confer cold and freeze tolerance in plants, harbor RNA chaperone activity [8,10,13]. A close link between RNA chaperone activity and cold or freeze tolerance has also been demonstrated in the study of GRP family members. Arabidopsis AtGRP7, which accelerates seed germination and seedling growth under low temperatures and confers freeze tolerance to Arabidopsis, harbors RNA chaperone activity [21,22], whereas AtGRP4, which does not affect seed germination or seedling growth under cold stress conditions [23], harbors no RNA chaperone activity [21]. Importantly, TaRZ2, OsRZ2, and AtGRP7, which harbor RNA chaperone activity, are localized to both the nucleus and cytoplasm (Figure 2) [10,22]. It is likely that RNA chaperone activity of RZs in the cytoplasm facilitates efficient translation at low temperatures in plants as observed in bacteria [17][18][19][20]. The mechanistic roles of RZs in plants under salt or dehydration stress conditions are not clearly understood. The expression of TaRZ1 and TaRZ2 was downregulated in wheat by salt stress (Figure 1), and seed germination and seedling growth of TaRZ1-or TaRZ2-expressing plants were retarded under salt stress conditions (Figures 3 and 4). Although the functional role of TaRZ1 and TaRZ2 in wheat under salt stress is not yet understood, these results suggest that downregulation of TaRZ1 and TaRZ2 should be an adaptive response of wheat to salt stress. It is likely that TaRZ1 and TaRZ2 exert their roles by either directly or indirectly controlling the transcript levels and/or RNA metabolism of target mRNAs that are negatively involved in salt stress response in wheat.
In conclusion, our results provide information for a better understanding of the different roles of TaRZ family members under various abiotic stresses. RZ family members in wheat, rice, and Arabidopsis are structurally conserved, and some of them are functionally conserved by acting as RNA chaperones under cold or freezing stress conditions. Future efforts are needed to  determine functional roles of RZs in wheat under stress conditions. We anticipate that the findings described here will be useful in future studies directed at understanding the functional roles of RZs in the response of wheat to diverse abiotic stresses. Considering that RZ family members should exert their functions by interacting with RNAs, searching for the target RNAs for specific RZs would also be important for a much deeper understanding of the cellular roles and molecular mechanisms of RZs in both monocotyledonous and dicotyledonous plants under stress conditions.

Plant materials, stress treatments, and expression analysis
The winter wheat used in this study was a T. aestivum cv. Keum Kang variety which is cultivated in Korea. Wheat plants were grown in soil at 23uC under 16 h light/8 h dark photocycle. For salt or dehydration stress treatment, 2-week-old wheat seedlings were submerged into a solution containing 300 mM NaCl or 250 mM mannitol, respectively. For cold stress treatment, 2-weekold seedlings were placed in a growth chamber at 4uC. The samples were collected at the indicated time intervals, and total RNA was extracted from the frozen plant samples using the Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). Transcript levels of each gene were determined via real-time RT-PCR with the gene-specific primers listed in Table 1. All experimental conditions for real-time RT-PCR and data analysis were essentially as described previously [8]. Briefly, real-time quantification of the RNA transcripts was performed in the Rotor-Gene Q thermal cycling system (Qiagen) using QuantiTect SYBR Green RT-PCR kit (Qiagen). The reaction mixture (25 mL) contained 200 ng of total RNA, 0.5 mM of each primer listed in Table 1, and appropriate amounts of enzymes and fluorescent dyes as recommended by the manufacturer (Qiagen). Relative expression levels of each gene were calculated after normalization of the transcript abundance using Actin gene as a reference. All experiments were repeated at least three times.

Vector construction and Arabidopsis transformation
To generate transgenic Arabidopsis plants expressing each TaRZ, the full-length cDNA of TaRZs was cloned into the BamHI/SalI site of the pCAMBIA 1301 vector, which expresses each TaRZ under control of the CaMV 35S promoter. Arabidopsis transformation was performed according to the vacuum infiltration method [24] using Agrobacterium tumefaciens GV3101. To identify transgenic plants, seeds were harvested and plated on selection medium containing hygromycin (50 mg ml 21 ) and carbenicillin (250 mg ml 21 ). Expression of each TaRZ in the transgenic plants was confirmed by RT-PCR analysis. The T 3 or T 4 homozygous lines were selected and used for phenotypic investigation.

Analysis of cellular localization of TaRZ proteins
To determine cellular localization of TaRZ proteins, the cDNAs corresponding to each TaRZ protein were fused in-frame with GFP. The TaRZ-GFP fusion protein was transiently expressed in tobacco plants under control of the CaMV 35S promoter [25]. Leaf samples were mounted on microscope slides, and the cellular expression of TaRZ-GFP proteins was observed under a confocal microscope (Carl Zeiss, Inc. Thornwood, NY, USA). Excitation and emission wavelengths were 488 and 505-545 nm, respectively. To confirm ER localization of TaRZ2 protein, the tobacco leaves were co-transformed with microsomal delta-12 fatty acid desaturase (FAD2) that is localized to the ER and catalyzes the first committed step of the biosynthesis of polyunsaturated fatty acids [14], and the signals in ER were observed under a confocal microscope.

Germination and seedling growth assays under abiotic stress conditions
A. thaliana (Col-0 ecotype) wild-type and transgenic plants were grown in a growth chamber at 23uC under long day conditions (16 h light/8 h dark). Seed germination and seedling growth under stress conditions were conducted essentially as described previously [22,26]. Sterilized seeds were sown on half-strength Murashige-Skoog (MS) medium supplemented with 75-175 mM NaCl or 200-300 mM mannitol for salt or dehydration stress treatments, respectively. For low temperature stress treatment, seeds on MS medium were kept in a growth chamber maintained at 10uC. 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 3day-old seedlings were transferred to MS medium supplemented with NaCl or mannitol. To determine the effect of cold stress on seedling growth, 3-day-old seedlings germinated under normal conditions were placed in a growth chamber maintained at 10uC.

Freeze tolerance and electrolyte leakage tests
Both non cold-acclimated (NA) and cold-acclimated (CA) freeze tolerance tests were conducted using 21-day-old wild-type and transgenic plants grown in pots. For the NA freeze tolerance tests, the plants were placed at 26uC for 3-6 h directly under continuous light. For the CA freeze tolerance tests, the plates were first placed at 4uC for 1 day and then subjected to a series of temperature treatments; 21uC for1 day, 27uC for 12 h, 16 h and 25 h under continuous light. After freezing shock, the plants were immediately placed at 4uC for 1 day in the dark, and then in a growth chamber under normal conditions. The electrolyte leakage test was conducted essentially as previously described [22]. In brief, the leaves from 3-week-old Arabidopsis plants were placed in a test tube containing 100 ml distilled water, and the tube was placed at 0uC for 1 h. An ice crystal was added to the tube, and the temperature of the water bath was decreased to 210uC at a rate of 1uC per 30 min. The tubes were removed from the water bath at the indicated temperatures, and the conductivity of the solution was measured before and after autoclaving for 10 min at 121uC. The ratio of electrolyte content before and after autoclaving was utilized as an indicator for membrane damage. The experiment was repeated at least four times.