In our previous study, 187 bZIP transcription factor family members were identified from the wheat genome (Li X. et al., 2015). Among these, 35 novel TabZIP proteins belonged to Subgroup A of the wheat bZIP family. To obtain the full-length coding sequences (CDSs) of these Subgroup-A TabZIP genes, their CDSs were used as queries to perform similarity search in the Triticeae full-length CDS database (TriFLDB; http://trifldb.psc.riken.jp/v3/index.pl; Mochida et al., 2009).

Multiple protein sequence alignment of the Subgroup-A members of the bZIP family in wheat, Arabidopsis and rice, including 41 TabZIP, 13 AtbZIP, and 17 OsbZIP proteins, respectively, was performed using the ClustalX program (Version 2.1) with the default settings (Larkin et al., 2007). An unrooted phylogenetic tree was constructed with the neighbor-joining method using MEGA5.0 software (Tamura et al., 2011).

Protein sequences of TabZIPs, AtbZIPs, and OsbZIPs were searched for common conserved motifs using the online tool MEME (Version 4.10.2, http://meme-suite.org/tools/meme; Bailey et al., 2009), and the number of motifs was specified with all other parameters set to default.

GSDS V2.0 (http://gsds.cbi.pku.edu.cn/; Hu et al., 2015) was used to align the CDSs of the Subgroup-A TabZIP genes with their corresponding genomic sequences to analyze their exon–intron structures.

To further analyze the intron-position patterns within the bZIP domains, protein sequences of the bZIP domains of the Subgroup-A TabZIP proteins were extracted using ScanProsite (http://prosite.expasy.org/; de Castro et al., 2006).

To identify homologous genes within Subgroup A of the wheat bZIP family, pairwise protein sequence alignments were performed using the BLASTP 2.3.0+ program (Altschul et al., 2005). A pair of homologous genes was defined based on the following criteria: (1) the FASTA-aligned region between their protein sequences covered ≥70% of the longer sequence and (2) the identity of the aligned region was ≥75% (Gu et al., 2002).

The wheat (T. aestivum L.) genotype “Luohan 9769” was used in this study. After sterilization with 75% ethanol and washing with sterilized water, wheat seeds were germinated and cultivated with double-distilled water in a growth chamber (25°C, 500 μmol/m²/s, 12/12 h light/dark photoperiod). Twelve-day-old wheat seedlings were treated with 15% (w/v) polyethylene glycol-6000 (PEG-6000), 250 mM NaCl, 200 μM ABA, or low temperature (4°C). The treated plants were stressed in the PEG or NaCl solutions for 24 h, cultivated under low temperature conditions for 24 h or sprayed once with the ABA solution. Three entire wheat seedlings were harvested as a pooled sample at 0, 1, 3, 6, 12, and 24 h after treatment, respectively.

To investigate the tissue-specific expression pattern of the gene TabZIP174 in wheat, roots, and leaves of 12-day-old seedlings, as well as roots, stems, leaves, and anthers at the blooming stage, were harvested. At harvest, plant materials were frozen immediately in liquid nitrogen and stored at −80°C.

Total RNA extraction and first-strand cDNA synthesis were performed as described previously (Li X. et al., 2015). The qRT-PCR analysis was performed using an Eco Real-Time PCR system (Illumina, San Diego, CA, USA) with TaKaRa SYBR® Premix Ex Taq™ (Tli RNaseH Plus) (TaKaRa, Dalian, China). The wheat Actin (Gene ID: 542814) and Arabidopsis Actin2 (AT3G18780) were used as the internal controls for the expression analysis of TabZIP genes in wheat and stress-responsive genes in Arabidopsis, respectively. The gene-specific primers were designed using Primer Premier 5.0 (Singh et al., 1998). Each reaction was performed in triplicate in a reaction volume of 10 μl as described previously (Li X. et al., 2015). The expression analysis was conducted using the same PCR procedure or with a slightly adjusted annealing temperature. The relative gene expression levels were calculated according to the 2^−ΔΔC_T method (Livak and Schmittgen, 2001).

Nuclear localization signal (NLS) prediction for the Subgroup-A TabZIP proteins was performed with the online tool NucPred (http://www.sbc.su.se/~maccallr/nucpred/; Brameier et al., 2007). To further examine the subcellular localization of the Subgroup-A TabZIP proteins, green fluorescent protein (GFP) expression vectors (CaMV35S-GFP-NOS) were constructed. The coding regions of TabZIP9, TabZIP27, TabZIP74, TabZIP138, and TabZIP174 were amplified by PCR with gene-specific primers and independently fused to the N-terminus of GFP in the expression vector. Wheat protoplasts were isolated from the mesophyll tissue of 2-week-old wheat seedlings, and then transformed using the PEG transfection method separately with the plasmid DNA of 35S::TabZIP9-GFP, 35S::TabZIP27-GFP, 35S::TabZIP74-GFP, 35S::TabZIP138-GFP, 35S::TabZIP174-GFP, and 35S::GFP control as described previously (Shan et al., 2014). After PEG transfection, wheat protoplasts were incubated in W5 solution (2 mM MES, 154 mM NaCl, 125 mM CaCl₂, and 5 mM KCl, pH = 5.7) in a dark chamber at 23°C for 18 h, and GFP fluorescence was monitored under a laser-scanning confocal microscope (A1, Nikon Corporation, Tokyo, Japan).

The full-length opening reading frame of TabZIP174 was amplified from wheat cDNA with gene-specific primers (forward: 5′-ATGGAGATGCCGGGAGGGA-3′; reverse: 5′-CTACCACGGACCCGTCAGAGTTC-3′), and cloned into the pBI121 vector driven by cauliflower mosaic virus (CaMV) 35S promoter to construct the recombinant vector (35S::TabZIP174). The recombinant vector (35S::TabZIP174) was introduced into Agrobacterium tumefaciens and transformed into Arabidopsis (ecotype Columbia-4) using the floral dip method (Zhang et al., 2006). Positive transgenic lines were screened on Murashige and Skoog (MS) medium containing 50 μg/ml kanamycin, and then confirmed by reverse transcription PCR. Independent T₃-generation transgenic Arabidopsis lines with relatively higher TabZIP174 transcript levels were chosen for further analyses.

Homozygous T₃ seeds of TabZIP174 transgenic lines were used for germination and primary root growth assays. Homozygous T₃ transgenic and wild type (WT) seeds were surface-sterilized, kept at 4°C in the dark for 3 days and then sown on ½ MS medium solidified with 1.0% (w/v) agar. For the germination assay, the seeds of transgenic lines and WT were placed on ½ MS medium containing no or 5% (w/v) PEG-6000. The germination percentages were calculated daily for 7 days. For the primary root growth assay, 3-day-old seedlings grown on ½ MS medium were transferred to ½ MS medium supplemented with or without 5% PEG-6000, and grown vertically for 4 days prior to measuring primary root lengths.

Homozygous T₃-generation TabZIP174 transgenic lines were used in the drought tolerance assay. Both transgenic and WT seeds were kept at 4°C in the dark for 3 days and then germinated in a soil mixture (1:1 of vermiculite:humus) in a greenhouse (22°C, relative humidity 70%, and 12/12 h light/dark photoperiod). Two-week-old seedlings were transferred to identical rectangular pots filled with the soil mixture and were regularly watered for two weeks. Subsequently, transgenic and WT plants were cultivated without watering for 4 weeks, and were then rewatered. Survival rates were calculated at 4 days after rewatering. The drought tolerance experiment was carried out in triplicate.

To determine the water loss rate under water deficit conditions, the aboveground parts were excised from 4-week-old transgenic and WT plants, and weighted immediately (fresh weight, FW). The samples were then placed on the laboratory bench (22–24°C, relative humidity 40–45%) and weighted at the designated time-points. The samples were finally oven dried at 80°C for 24 h to a constant dry weight (DW) (Mao et al., 2012). The percentages of water loss were measured relative to the initial water contents. Subsequently, relative water contents (RWCs) before oven drying were also calculated. Ten plants for each of transgenic lines and WT were used in this assay.

Arabidopsis leaves were harvested at designated time-points during drought treatment and used to measure the free proline content, total soluble sugar content, and osmotic potential. To maximize the sample uniformity at each time-point, leaves of the same size and location were detached from transgenic and WT plants. The free proline content was determined as described previously (Bates et al., 1973). Samples (~0.1 g) were homogenized in 3% sulfosalicylic acid and boiled for 10 min. After the reaction between proline and acid ninhydrin, the absorbance of sample solutions was measured at 520 nm with a UV-Vis spectrophotometer (NanoDrop 2000c, Thermo Scientific, Wilmington, DE, USA). The total soluble sugar content was measured using the anthrone colorimetric method (Yemm and Willis, 1954). Soluble sugars were extracted from homogenized samples (~0.1 g). After the reaction of soluble sugars with anthrone and concentrated sulfuric acid, the absorbance of sample solutions was measured at 620 nm. The proline and soluble sugar contents were determined using their respective standard curves and calculated based on fresh weights. Osmotic potential was measured with an automatic freezing-point osmometer (Multi-OSMETTE™, Model 2430E, Precision Systems Inc., Natick, MA, USA). Ten leaves per Arabidopsis line were harvested as a pooled sample and transferred to an injection syringe, and the liquid was then squeezed out of the leaves. The supernatant tissue sap was collected after centrifugation at 13,400 × g for 1 min at room temperature, and then filtered with a filtration column. The filtrate was loaded onto the osmometer to measure the osmotic concentration according to the manufacturer's instructions. The osmotic concentration was measured three times for each sample. Subsequently, the osmotic potential was calculated from the osmotic concentration using the Van't Hoff equation. All of these measurements were repeated three times.

The total chlorophyll content was determined with a portable chlorophyll meter (SPAD-502Plus, Konica Minolta Inc., Tokyo, Japan). Thirty leaves of similar size were selected from each Arabidopsis line and used for the in situ measurement of the chlorophyll content at each designated time-point during the drought treatment. To minimize the measurement deviations of different leaves, the chlorophyll meter was applied to the same position on the leaves.

In our previous study, 187 wheat bZIP transcription factor genes were identified from the wheat genome and named based on their chromosomal locations (Li X. et al., 2015). And 35 novel TabZIP, 13 AtbZIP, and 17 OsbZIP genes were identified as Subgroup-A members of the bZIP family based on the phylogenetic analysis (Li X. et al., 2015). To obtain the full-length CDSs of the 35 Subgroup-A TabZIP genes, the Triticeae full-length CDS database (TriFLDB) (Mochida et al., 2009) was used to perform similarity search with the CDSs of these 35 TabZIP genes as queries. The basic information on these 35 TabZIP genes is shown in Table 1. In addition to these 35 novel TabZIP genes, Subgroup A of the wheat bZIP family included TaABF1, TaABI5, WABI5 (WABI5-1, WABI5-2, and WABI5-3), TaABL1, and TabZIP60 (TabZIP60-A, TabZIP60-B, and TabZIP60-D) (Johnson et al., 2002, 2008; Kobayashi et al., 2008; Rikiishi et al., 2010; Harris et al., 2013; Xu et al., 2014; Zhang et al., 2015). Therein, TaABF1, WABI5-3, and TabZIP60-A were identical to TabZIP23, TabZIP162, and TabZIP51 in our nomenclature, respectively. In conclusion, a total of 41 Subgroup-A members of the bZIP family were identified from the wheat genome.

Multiple protein sequence alignment of the Subgroup-A members of the bZIP family in wheat, Arabidopsis, and rice (including 41 TabZIP, 13 AtbZIP, and 17 OsbZIP proteins) was performed, and then, an unrooted phylogenetic tree was constructed (Figure 1). A majority (9/13) of the AtbZIPs in the phylogenetic tree had been characterized in previous studies (Menkens and Cashmore, 1994; Choi et al., 2000; Finkelstein and Lynch, 2000; Uno et al., 2000; Lopez-Molina et al., 2001; Bensmihen et al., 2002, 2005; Carles et al., 2002; Kang et al., 2002; Kim et al., 2002, 2004; Lopez-Molina et al., 2002; Finkelstein et al., 2005; Yoshida et al., 2010; Mendes et al., 2013), and three of the nine AtbZIPs played important roles in regulating abiotic stress resistance in Arabidopsis (Choi et al., 2000; Kang et al., 2002; Kim et al., 2004; Finkelstein et al., 2005). Additionally, 5 of the 17 OsbZIPs in the phylogenetic tree had important functions in enhancing tolerance of transgenic rice to multiple abiotic stresses (Xiang et al., 2008; Zou et al., 2008; Lu et al., 2009; Hossain et al., 2010a,b; Tang et al., 2012). Among the 41 TabZIPs of Subgroup A, only WABI5-1, TaABL1, and TabZIP60 were involved in regulating plant responses to abiotic stress (Kobayashi et al., 2008; Xu et al., 2014; Zhang et al., 2015). Clade A1 in the phylogenetic tree comprises 20 members, of which 75% (15/20) were functionally characterized, with the ABFs (ABF1–4) among this subset of proteins. On the other hand, most (13/15) of the bZIP proteins previously known to participate in the regulation of abiotic stress responses clustered in Clade A1, indicating that the four other unknown wheat bZIP proteins in Clade A1 (TabZIP40, TabZIP111, TabZIP171, and TabZIP174) were more likely associated with abiotic stress responses.

Common conserved motifs in the Subgroup-A proteins of the bZIP family in wheat, Arabidopsis and rice were also analyzed (Supplemental Figure 1). Most (9/11) of the Subgroup-A bZIP proteins related to abiotic stress responses (namely, AtbZIP36, AtbZIP37, AtbZIP38, OsbZIP23, OsbZIP46, OsbZIP72, WABI5-1, TaABL1, and TabZIP60) shared a majority of conserved motifs (Supplemental Figure 1). And, notably, TabZIP111 and TabZIP174 shared most of the conserved motifs with the above-mentioned bZIP proteins. Therefore, protein sequence similarity of this subset of proteins, which shared most of the conserved motifs, was further analyzed (Figure 2). These 12 Subgroup-A bZIP proteins had six conserved motifs in common, and the protein sequence of each motif was highly conserved among these bZIP proteins (Figure 2). Nine of the 12 bZIP proteins (AtbZIP36, AtbZIP37, AtbZIP38, OsbZIP23, OsbZIP46, OsbZIP72, WABI5-1, TaABL1, and TabZIP60) played important roles in plant responses to abiotic stress (Choi et al., 2000; Kang et al., 2002; Kim et al., 2004; Finkelstein et al., 2005; Kobayashi et al., 2008; Xiang et al., 2008; Lu et al., 2009; Hossain et al., 2010a; Tang et al., 2012; Xu et al., 2014; Zhang et al., 2015). The phylogenetic analysis and similarity in the conserved motifs indicated that TabZIP111 and TabZIP174 were possibly involved in abiotic stress responses.

To obtain a deeper insight into the structures of the 35 novel TabZIP genes in Subgroup A, we mapped their exon/intron organizations. Only two (5.7%) of these genes were intronless, and the others had 1–3 introns (Figure 3). Specifically, 10 TabZIP genes had three introns, 15 had two introns, and 8 had one intron.

Not surprisingly, neighboring TabZIP genes in the phylogenetic tree tended to share similar gene structures (Figure 3). TabZIP9 and TabZIP68 possessed identical exon/intron organizations, as did TabZIP23/TabZIP80, TabZIP114/TabZIP158, and TabZIP14/TabZIP133/TabZIP134. Except slight variations in the first exon lengths, TabZIP5 and TabZIP65, as well as TabZIP27, TabZIP90, and TabZIP148, had nearly identical exon/intron compositions. Similarly, TabZIP80 differed from TabZIP86 only by 12 nucleotides (nt) in the last exon lengths. Both TabZIP73 and TabZIP135 had only one exon, despite a difference in exon length. Differences in exon lengths between these homologous TabZIP genes (Table 2) could potentially lead to their functional divergences.

In addition, for 30 (85.7%) of these 35 Subgroup-A TabZIP genes, the first exon was longer than the subsequent exons in the CDS. Statistical analysis showed that exons of 72, 81, 30, and 93 nt in length appeared frequently within the CDSs of the 35 Subgroup-A TabZIP genes, with 23 (72 nt-exon), 13 (81 nt), 10 (30 nt), and 6 (93 nt) occurrences, respectively. Moreover, the exon lengths of the Subgroup-A TabZIP genes were all multiples of three without exception (Figure 3), demonstrating that all of their introns were in Phase 0 (P0).

The intron positions within the bZIP domains were also analyzed. Among the 33 intron-containing TabZIP genes, 32 had introns within the bZIP domain region. The 35 Subgroup-A TabZIP genes were divided into seven patterns (a–g) (Supplementary Figure 2), based on intron numbers and positions, which was consistent with previous studies in rice and maize (Nijhawan et al., 2008; Wei et al., 2012).

A majority (57.1%) of the Subgroup-A TabZIP genes were of Pattern a, and these TabZIP genes possessed only one intron, which was in P0 and located between the codons encoding the amino acids Gln and Ala (or Ser) in the hinge region (Supplementary Figures 2, 3). This was in accordance with previous studies (Nijhawan et al., 2008; Wei et al., 2012). Patterns b and d were similar in that both had two introns in P0, one within the hinge region and the other within the leucine zipper region. The only difference was that the intron within the leucine zipper region was inserted between Gln at Position +19 and Ala (or Lys) at Position +20 in Pattern b, but was inserted between Glu at Position +21 and Leu at Position +22 in Pattern d. Pattern e had three introns, one in the hinge region and two in the leucine zipper region. Two of them shared the same positions with the two introns of Pattern b, one intervening between the amino acids at Positions −5 and −6 in the hinge region and the other between the amino acids at Positions +19 and +20 in the leucine zipper region. Pattern f had only one intron within the leucine zipper region, and was only found in one TabZIP gene. Pattern g lacked any intron in the bZIP domain region and was found in three TabZIP genes.

Notably, if present, introns within the hinge region always occurred at the same position, between Gln (or Arg) at Position −6 and Ala (or Ser) at Position −5 (such as Patterns a, b, d, and e), which was in agreement with the findings of a previous study (Nijhawan et al., 2008). However, introns present in the leucine zipper region were at variable positions. In our case, no introns were within the basic region of the bZIP domain. To sum up, TabZIP genes with close phylogenetic relationships tended to share similar exon/intron organizations, and the positions of the introns within the hinge regions of the bZIP domains remained well conserved during the evolution of the Subgroup-A TabZIP genes.

As homologous genes typically retain similar biological roles, identifying homologous genes is important for transferring functional information between genes (Remm et al., 2001). To identify homologous genes within Subgroup A of the wheat bZIP family, pairwise protein sequence alignments were performed using the BLASTP 2.3.0+ program (Altschul et al., 2005). A total of 13 groups of homologous bZIP genes were identified from Subgroup A and included two categories: homologs (11 groups) and paralogs (2 groups) (Table 2).

Modern wheat (T. aestivum, 2n = 6x = 42, AABBDD) derives from two hybridizations between three gramineous ancestors (Brenchley et al., 2012; Jia et al., 2013). The wheat genome is composed of A, B, and D sub-genomes. In wheat, homologous genes located at similar positions on three corresponding chromosomes from the A, B, and D sub-genomes are generally referred to as homologs. For instance, TabZIP14, TabZIP74, and TabZIP133 were three homologs located at similar positions on the long arms of chromosomes 2A, 2B, and 2D, respectively (Figure 4). They shared high similarities in protein sequences with the aligned region between each pair covering the whole sequences, and their identities were 92, 98, and 94%, respectively.

Tandem paralogs are homologous genes located at neighboring positions of the same chromosome. In this study, we identified two groups of tandem paralogs within Subgroup A of the wheat bZIP family, TabZIP133/TabZIP134 and TabZIP80/TabZIP84/TabZIP86 (Table 2). Paralogs TabZIP133 and TabZIP134 possessed identical protein sequences and were adjacent to each other in the long arm of chromosome 2D (Figure 4), indicating that this pair of paralogs probably derived from one tandem duplication event, which often led to the generation of paralogs. Paralogs TabZIP80, TabZIP84, and TabZIP86 were all located on chromosome 3B and shared high similarities in protein sequences. Specifically, the aligned region between TabZIP80 (391 aa) and TabZIP86 (387 aa) covered their entire sequences, and the identity within the aligned region was 89%. The aligned region between TabZIP80 and TabZIP84 (286 aa) covered the entire TabZIP84 sequence and 73% of the TabZIP80 sequence, with an identity of 90%. The aligned region between TabZIP84 and TabZIP86 covered the entire TabZIP84 sequence and 74% of the TabZIP86 sequence, with an identity of 87%. This suggested that this paralogous group (TabZIP80/TabZIP84/TabZIP86) might have been derived from two tandem duplication events. Further genomic sequence alignment among them showed that the unspliced transcript sequence of TabZIP84 (including 5′- and 3′-untranslated regions, two exons and one intron) was aligned with only a portion of the unspliced transcript sequence of TabZIP80 (or TabZIP86) (data not shown), indicating that the gene TabZIP84 derived from only a segment of TabZIP80 (or TabZIP86).

Compelling evidence demonstrated that the expression of Subgroup-A bZIP genes in Arabidopsis and rice was induced by ABA and various abiotic stress treatments (Choi et al., 2000; Xiang et al., 2008; Hossain et al., 2010b; Tang et al., 2012). To further confirm whether the expression of Subgroup-A bZIP genes in wheat was induced by different abiotic stresses, quantitative real-time PCR (qRT-PCR) was performed to analyze the expression profiles of these TabZIP genes following PEG, NaCl, cold (4°C) or ABA treatments. Overall, the expression of 28 of these 35 Subgroup-A TabZIP genes responded to all four treatments and exhibited complicated variation trends following the treatments (Figure 5).

Among the expression profiles of the 28 TabZIP genes under the four different treatments (28 × 4 = 112), 20 exhibited a variation trend in which the transcript level gradually ascended and reached a maximum, followed by a continuous decline. These included TabZIP5 (under PEG treatment), TabZIP9 (cold), TabZIP20 (cold), TabZIP40 (NaCl, cold or ABA), TabZIP51 (cold), TabZIP65 (cold), TabZIP76 (PEG), TabZIP77 (cold), TabZIP83 (cold), TabZIP114 (PEG or cold), TabZIP127 (ABA), TabZIP133 (cold), TabZIP138 (cold), TabZIP158 (PEG or ABA), TabZIP171 (cold), and TabZIP174 (cold).

In addition, 10 other expression profiles had an inverse variation trend in which the transcripts decreased to the lowest level and then increased. These included TabZIP14 (NaCl or cold), TabZIP74 (PEG or ABA), TabZIP77 (ABA), TabZIP80 (cold), TabZIP133 (PEG or NaCl), and TabZIP174 (PEG or NaCl).

Under the NaCl treatment, the expression level of TabZIP86 continuously increased and reached its maximum at 24 h after treatment. In contrast, for TabZIP27 (PEG) and TabZIP74 (cold), the transcript levels declined continuously until reaching their lowest levels at 24 h after treatment. After the 24-h PEG treatment, the expression level of TabZIP27 was only one-seventh of that before treatment. The transcript level of TabZIP74 after the 24-h cold treatment dropped to one-tenth of that before treatment. Thus, the expression levels of TabZIP27 and TabZIP74 were down-regulated by PEG and cold treatments, respectively.

Notably, the expression of several TabZIP genes (such as TabZIP40, TabZIP76, and TabZIP111) responded rapidly to the ABA treatment because their expression levels increased rapidly immediately after a 1-h exposure to exogenous ABA. For instance, the transcript level of TabZIP76 was more than 10 times that before treatment, while the transcript levels of TabZIP40 and TabZIP111 were ~6.5 and 7 times those before treatment, respectively.

Interestingly, some homologs (or paralogs) exhibited similar expression profiles following certain treatments, such as TabZIP5/TabZIP65 (cold), TabZIP14/TabZIP133 (NaCl), TabZIP74/TabZIP133 (PEG), TabZIP27/TabZIP90 (PEG), TabZIP27/TabZIP90/TabZIP148 (cold), TabZIP27/TabZIP148 (NaCl or ABA), TabZIP83/TabZIP138 (cold), and TabZIP114/TabZIP158 (PEG). However, most homologous TabZIP genes were expressed differently under the same treatments (Figure 5). Indeed, TabZIP80 and TabZIP86 even displayed inverse variation trends of expression under the NaCl (or cold) treatment. After the NaCl treatment, TabZIP86 transcripts accumulated gradually and reached a maximum at 24 h post-treatment, while, in contrast, TabZIP80 expression was down-regulated by NaCl, with its transcript level dropping to a relatively lower level after the 24-h treatment. This suggested that homologous TabZIP genes in Subgroup A had undergone expression pattern shifts.

Our phylogenetic and conserved motif analyses suggested that TabZIP174 (belonging to Clade A1) were more likely involved in abiotic stress response. In addition to TabZIP174, several Subgroup-A TabZIP members (TabZIP9, TabZIP27, TabZIP74, and TabZIP138) were selected from other clades to confirm their subcellular localization. First, the NLSs were predicted using their protein sequences. A NLS, consisting of 4–8 amino acid (e.g., Pro, Lys, or Arg) residues, commonly exists within a transcription factor protein (Kalderon et al., 1984). The basic region within the bZIP domain generally includes a NLS followed by an invariant motif (N-X₇-R/K) that is responsible for contacting the DNA (Jakoby et al., 2002). According to the NLS prediction result by NucPred (Brameier et al., 2007), TabZIP27 possessed a NLS sequence (RRKKR) positioned three amino acid residues ahead of the N-terminus of the N-X₇-R/K motif, and TabZIP138 also had a NLS sequence (GRRKR) located away from the N-terminus of the bZIP domain.

To further examine the subcellular localizations of these five TabZIP proteins, wheat protoplasts were separated from mesophyll tissue and then transformed with 35S::TabZIP9-GFP, 35S::TabZIP27-GFP, 35S::TabZIP74-GFP, 35S::TabZIP138-GFP, and 35S::TabZIP174-GFP fusion vectors, respectively. The 35S::GFP vector served as the control. Subsequently, GFP expression was monitored by confocal microscopy at 18 h after PEG transformation.

Green fluorescence was detected in the nucleus of wheat protoplasts for TabZIP27-GFP, TabZIP74-GFP, TabZIP138-GFP, and TabZIP174-GFP fusion proteins (Figure 6), indicating that TabZIP27, TabZIP74, TabZIP138, and TabZIP174 proteins were exclusively localized in the nucleus. Nevertheless, green fluorescence of TabZIP9-GFP fusion protein was simultaneously present in the nucleus, cytoplasm, and cell membrane.

The foregoing phylogenetic and conserved motif analyses suggested that TabZIP174 potentially participated in regulating abiotic stress response. To further investigate the role of TabZIP174 in response to abiotic stress, 35S::TabZIP174 transgenic Arabidopsis lines were generated. To examine differences in the germination rates and primary root growth between TabZIP174 transgenic and WT Arabidopsis, seeds of three transgenic lines (OE-2, OE-4, and OE-5) and WT were germinated on MS medium.

Under normal conditions, there were no significant differences in germination rates and primary root lengths between the transgenic lines (OE-2, OE-4, and OE-5) and WT. However, 3 days after the PEG treatment, the germination rates of the transgenic lines (OE-2, OE-4, and OE-5) were higher than that of WT (Figure 7A), and the differences between OE-2 and WT and between OE-4 and WT both reached the significant level (t-test, P < 0.05). Compared with WT, the germination rates of the transgenic lines OE-2, OE-4, and OE-5 increased by ~14, 17, and 12%, respectively.

In the presence of PEG, primary root elongation was significantly inhibited for both the transgenic lines and WT, however, the primary root lengths of lines OE-2, OE-4, and OE-5 were significantly greater than those of WT plants (t-test, P < 0.01) (Figures 7B,C). This indicated that the inhibitory effect of PEG on primary root growth was more serious in WT than in the transgenic plants. Thus, our results suggested that TabZIP174 overexpression enhanced the tolerance of transgenic Arabidopsis to the imposed drought stress.

Under well-watered conditions, there were no evident morphological differences between transgenic and WT Arabidopsis throughout their life cycles.

Four TabZIP174 transgenic lines were selected for testing in soil to characterize their performances under drought stress. At the early stage of the drought treatment (e.g., 2 weeks before rewatering), TabZIP174 transgenic and WT plants grew normally, with no notable phenotypic differences between them (Figure 8A: the upper row).

Eighteen days after withholding water, the lower rosette leaves of WT plants showed slight wilting, whereas TabZIP174 plants still grew normally. After 4-week drought treatment (just before rewatering), most WT plants were severely wilted and a number of rosette leaves were yellow or dead. In contrast, although most TabZIP174 plants were wilted and many rosette leaves were severely curled, most leaves remained green and only some TabZIP174 plants displayed symptoms of severe water deficit (Figure 8A: the middle row).

Four days after rewatering, 38–60% of the TabZIP174 plants had survived, whereas only 18% of the WT plants had survived (Figure 8A: the lower row, Figure 8B). The survival rates of lines OE-2, OE-4, and OE-5 were 42, 60, and 49%, respectively, which were much higher (t-test, P < 0.01) than that of WT (18%). Thus, the overexpression of TabZIP174 greatly improved drought resistance in transgenic Arabidopsis.

Water loss rate is an important index that has been used to evaluate the water status of plants under water deficit conditions (Mao et al., 2012). In the present study, the water loss rates of the detached rosettes for transgenic lines were lower than that of WT plants, and the final relative water contents of transgenic lines were significantly higher than that of WT (t-test, P < 0.01) (Figure 9), indicating that TabZIP174 transgenic plants had stronger water retention capacity.

To explore whether TabZIP174 overexpression influenced proline accumulation, the free proline contents in the transgenic and WT plants were measured. After drought treatment, the proline contents of the transgenic lines were higher than those of the WT plants. And the difference between each transgenic line (OE-2, OE-4, or OE-5) and WT reached the significant level (t-test, P < 0.01) whether 2 or 3 weeks after drought treatment (Figure 10A).

The contents of total soluble sugars were also measured in the transgenic and WT plants under drought stress to determine whether the enhanced drought tolerance of the transgenic plants was associated with soluble sugars. Under water deficit conditions, the soluble sugar contents obviously increased compared with those before drought treatment for both transgenic and WT plants, and notably, the soluble sugar contents of transgenic lines OE-2, OE-4, and OE-5 were significantly higher than that of WT 2 weeks after drought treatment (t-test, P < 0.01) (Figure 10B). The results suggested that TabZIP174 probably participated in proline and carbohydrate metabolism.

To retain a relatively stable intracellular environment under abiotic stress conditions, many plants decrease their intracellular osmotic potential by accumulating organic osmolytes (such as proline, mannitol, and glycine betaine) in cells (Zhang et al., 2011). Our result revealed that 2 weeks after drought treatment, the transgenic plants had lower osmotic potential than WT. In contrast, the osmotic potential was higher in the transgenic plants than in WT 3 weeks after drought treatment (Figure 10C). And whether 2 or 3 weeks after drought treatment, the difference between transgenic line OE-4 (or OE-5) and WT reached the significant level (t-test, P < 0.05). Compared with WT plants, TabZIP174 transgenic lines maintained a relatively stable osmotic potential under water deficit conditions.

Additionally, the leaf chlorophyll content was measured to further determine possible physiological differences between transgenic and WT plants. Two weeks after drought treatment, the rosette leaves of all of the transgenic and WT plants remained green (Figure 8A: the upper row); with no evident difference in the chlorophyll content (Figure 10D). Three weeks after drought treatment, however, transgenic lines OE-4 and OE-5 had significantly higher chlorophyll contents than WT (t-test, P < 0.05) (Figure 10D). The severe drought resulted in a more rapid decrease in the chlorophyll content of WT than transgenic plants. This suggested that the overexpression of TabZIP174 could directly or indirectly slow chlorophyll degradation in leaves of transgenic plants under severe drought conditions.

Our results of phenotypic studies showed that TabZIP174 transgenic lines had improved resistance to drought stress. To further explore the underlying molecular mechanisms, we performed the expression analyses of nine stress-response-related genes in transgenic and WT plants under normal and water deficit conditions. Transcript levels of six genes (RD29A, RD29B, RAB18, DREB2A, COR15A, and COR47) were similar between transgenic lines and WT under normal conditions, whereas after PEG treatment, their transcript levels were significantly higher in transgenic lines than in WT (t-test, P < 0.05 or P < 0.01) (Figure 11). However, there were no significant differences in transcript levels of the other three genes (P5CS, ABI1, and ABI2) between transgenic lines and WT (data not shown).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.