A drought tolerant triticale cultivar., “Xinxiaoheimai 3” (developed and kindly provided by the Wheat Crop Research Institute of Shihezi University, Xinjiang, China; Li et al., 2008) was used for this study. After cleaning and disinfection, the seeds were planted in an arid lot with irrigation at the Experimental Station of the Agricultural College of Shihezi University (85° 59′ 50″ N, 44° 18′ 58″ E, Xinjiang, China). The experiment station is located at an altitude about 437–450 m with an annual rainfall of 208 mm, on average, and an evaporation capacity of about 1,660 mm, a typical continental climate suitable for irrigated agriculture. The soil type was calcaric fluvisol, a sandy loam soil texture with 19.5% clay, 32.1% silt, and 38.5% sand. The soil bulk (0–20-cm depth) property was characterized as 1.34 g cm⁻³ in density with 15.3 g kg⁻¹ organic matter, 1.0 g kg⁻¹ total N, 19.3 mg kg⁻¹ Olsen P and 194 mg kg⁻¹ total K.

To eliminate the effects of rainfall, a rain shelter was built over the drought stress plots. Plants flowering at the same time were selected during the flowering stage. In drought-stressed plots, irrigation was stopped when the triticale developed at the heading stage, and the control was irrigated normally. The plots were laid out randomly with three replications in both irrigated and drought-stressed areas. Proline, MDA and electrical conductivity levels in the plant leaves were assessed daily as described previously (Chen and Wang, 2002). Roots, stems, flag leaves, and immature grains of both the stressed and control plants were collected at a point when the proline, MDA and electrical conductivity increased (67.30%, 42.12%, and 37.67%, respectively) significantly, and the soil moisture content decreased (37.32%) significantly in the stressed plots.

RNA was extracted from the above-mentioned triticale leaves using Hipure HP Plant RNA Mini Kits (Magen). Using these RNA as templates, we performed reverse transcriptase PCR (RT-PCR) to synthesize cDNA sequences using SMARTScribe genome Reverse Transcriptase (TaKaRa, Dalian, China). Synthesized cDNA was stored at −20°C before being sequenced. Based on RNA-Seq sequencing results, the sequence of Unigene c51971 (708 bp) was studied in this research, and specific primers for 5′- and 3′-RACE using SMARTer RACE kits (Supplementary Table S1) were designed. The RACE procedure was as follows: 94°C for 2 min, 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 72°C for 35 min, and 16°C for 10 min. The PCR products were recovered and purified using 1.0% agarose gel electrophoresis. The purified PCR products were ligated to the pMD19-T vector (TaKaRa, Dalian, China) and transformed into TOPO10 cells (TIANGEN, China). Positive clones were identified and sequenced. The open reading frame (ORF) of the full-length cDNA sequence was obtained by splicing the sequencing results using an online tool CAP3¹ (Shang et al., 2019) and the National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI-BLAST). Sequence alignment analysis preliminarily identified the sequence as a triticale NAC gene, which was preliminarily designated TwNAC01.

The NCBI ORF Finder² was used to identify sequences homologous to the ORF and coding sequences of TwNAC01 (GenBank: MG736919). Conserved structures in the TwNAC01 gene were identified using Smart³ and the ProtParam tool.⁴ Physical and chemical properties of the predicted TwNAC01 protein were analyzed, as well as its hydrophobicity⁵ using the ExPASy server.⁶ Multi-alignment of TwNAC01 with other NAC proteins in different species was conducted using DNAMAN. Subcellular location of the protein was predicted using Protcomp and TargetP 1.1.⁷ The relevant sequences were aligned using MEGA (version 10.0; Shang et al., 2019) and MegAlign (DNAStar).

The RNA samples used for real-time fluorescence quantitative PCR (qRT-PCR) were from two sources, the drought-stressed triticale plants from stressed plots as mentioned above in plant materials, and seedlings treated with 20% PEG6000 (osmotic potential of about −0.50 MPa; Michel and Kaufmann, 1973; Michel et al., 1983), 200-mM NaCl, cold (4°C), 100-μM MeJA, or 100-μM ABA as follows (Huang et al., 2015). The triticale seedlings were cultured at 25°C with a normal watering regime, under a 12-h light/12-h dark cycle in a growth chamber. The seedlings were transplanted in hydroponic boxes when they had two leaves and one main shoot. Three hydroponic boxes were allocated to each treatment; each box had 12 holes with 5 seedlings in each hole. After transplantation, seedlings were allowed to acclimate for 5 days. After acclimation, one set of boxes was transferred to a cold room (4°C). Other boxes were treated with each of the followings: 1 L of 20% PEG6000 (osmotic potential of about −0.50 MPa; Michel and Kaufmann, 1973; Michel et al., 1983), 200-mM NaCl, 100-μM MeJA, and 100-μM ABA, respectively. In all treatments, plant roots were soaked and leaves were sprayed with the same solution. Roots and leaves were collected after 0, 1, 3, 6, 12, and 24 h of treatment. Collected samples were frozen in liquid nitrogen and then transferred to a freezer at −80°C. The relative expression levels of target genes were calculated with the 2^−ΔΔCt method (Livak and Schmittgen, 2001).

RNA was extracted from the triticale materials collected from each of the six treatments (field drought, 20% PEG6000, 200-mM NaCl, cold (4°C), 100-μM MeJA, and 100-μM ABA) using Hipure HP Plant RNA Mini Kits (Magen, China), and cDNA was synthesized using 5X All-ln-One RT MasterMix (Applied Biological Materials, Canada) with specific primers (qRT-PCR primer pair in Supplementary Table S1). The wheat actin gene, TaActin, was used as internal reference for real-time qPCR (Supplementary Table S1). qPCR was performed using SuperReal PreMixPlus (SYBR Green) kits (Tiangen, China). Each 10-μl qPCR volume contained 6 μl 2× SuperReal PreMixPlus, 0.25-μl forward primer, 0.25-μl reverse primer, 1-μl cDNA template, and sufficient ddH₂O to make 10 μl. qRT-PCR amplifications were performed using a Roche Light-Cycler 480R with the following cycling conditions: pre-denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 61°C for 30 s, and amplification at 72°C for 30 s. All reactions were performed in triplicate, and relative gene expression levels were determined using the 2^-ΔΔCt method (Livak and Schmittgen, 2001).

The coding sequence of the TwNAC01 gene was cloned into the plant subcellular expression vector pCAMBIA1301S Enhanced Green Fluorescent Protein (EGFP; GenBank accession E17099). Insertion primers containing BamHI-XbaI restriction sites (Supplementary Table S1) were designed and ligated using a ClonExpressII one-step cloning kit (TaKaRa, Dalian, China). After verification via sequencing, the recombinant plasmid and the empty vector control (VC) were transformed into Agrobacterium tumefaciens GV3101 (TaKaRa, Dalian, China; Mao et al., 2014). GV3101 carrying the recombinant plasmid or the empty vector (TaKaRa, Dalian, China) was cultured on Luria-Bertani (LB) medium containing Kan + and Rif+. When the OD600 of the bacterial solution reached 0.5–0.6, the bacterial solution was collected and re-suspended in infection buffer [10-mM MgCl₂, 10-mM fatty acid methyl ester sulfonate (MES), 150-μM surfactant-AS, pH 5.7].

Subcellular location was visualized in tobacco leaves. Tobacco seeds were planted in a growth chamber and cultured at 23°C, with 60% relative humidity and a 16-h light/8-h dark cycle, for 3 weeks prior to vector inoculation. The cultured A. tumefaciens solution was then injected into tobacco leaves with 5-ml needleless sterile syringe, and tobacco seedlings were then cultured in darkness for 36 h. Tobacco leaves exhibiting normal growth after inoculation were selected for examination. The area of each selected leaf around the infection site was excised. Enhanced Green Fluorescent Protein (EGFP) fluorescence signals in the tobacco leaves were observed using a Fluo-View confocal microscope (FV300, Olympus, Japan).

To obtain transgenic Arabidopsis plants, the coding sequence containing the stop codon of TwNAC01 was amplified by RT-PCR and cloned into the KpnI and XbaI restriction sites of the pCAMBIA1300-35S vector (TaKaRa, Dalian, China) under the control of the 35S promoter of the cauliflower mosaic virus (CMV). The primers containing the KpnI and XbaI restriction sites are listed in Supplementary Table S1. The recombinant vector pCAMBIA1300-35S-TwNAC01 and the empty vector pCAMBIA1300-35S-VC were introduced into A. tumefaciens strain GV3101 (TaKaRa, Dalian, China). Finally, transgenic Arabidopsis plants were generated using the A. tumefaciens-mediated floral dipping method (Clough and Bent, 1998). To generate homozygous progeny, T1 and T2 seeds were selected on kanamycin (50 mg l⁻¹) plates. T3 transgenic and WT plants of A. thaliana were watered once at the rosette stage and then subjected to drought stress for 25 days. RNA was then extracted from the leaves and roots of both transgenic and WT A. thaliana using Hipure HP Plant RNA Mini Kits (Magen, China). TwNAC01 gene expression levels were then measured using semi-quantitative analysis with gene-specific primers (Supplementary Table S1) following the same qPCR protocol. Representative lines overexpressing TwNAC01 were used for further analysis.

We then measured various stress-related physiological indexes in 35-day-old transgenic A. thaliana overexpressing TwNAC01, mock-transformed A. thaliana, and WT A. thaliana. Leaf relative water content (RWC) was determined following the methods of Flexas et al. (2006), leaf electrical conductivity was measured following Chen and Wang (2002), leaf MDA content was estimated following Chen and Wang (2002), and leaf H₂O₂ content was determined using an H₂O₂ measurement kit (China Nanjing Jiancheng Science and Technology Co., Ltd). The concentration of hydrogen peroxide was estimated according to a standard formulae as: H₂O₂ (mmol/gprot) = (OD_tissue − OD_blank)/(OD_standard − OD_blank) * standard concentration (163 mmol/l), where H₂O₂ (mmol/gprot) is the hydrogen peroxide concentration measured from the testing tissue, OD_tissue is the OD value measured from the testing tissue, OD_blank is the OD value measured from the blank control, and OD_standard is the OD value of the kit standard. We also determined the rate of water loss in leaves. Five rosette leaves from each group of A. thaliana plants (WT, VC, and the three TwNAC01-overexpression lines) were collected, transferred to filter paper, and placed in a constant temperature incubator at 25°C. Leaves were weighed every hour for 8 h and photographed at three time points (2, 5, and 8 h). Water loss was characterized based on weight loss and the degree of leaf curl. Water loss rate measurements were replicated six times. After growing for 55 days, plants of all A. thaliana lines were carefully removed from the nutrient soil and washed. The length of the main root system of each plant was measured.

Specific primers were designed for PCR amplification of silencing fragments based on the 3′-UTR region of the TwNAC01 gene (Supplementary Table S1), and the barley stripe mosaic virus (BSMV) vector was constructed using ligation-independent cloning (LIC) as previously described (Lee et al., 2015). The BSMV-γb vector was digested with the ApaI restriction enzyme and the vector skeleton was recovered. The PCR fragments were then treated with T4 DNA polymerase in a reaction mixture containing 1-mM deoxythymidine triphosphate (dTTP). The mixture was subjected to react for 30 min at room temperature. After the completion of the reaction, the system was heated to 75°C for 10 min to inactivate the T4 DNA polymerase. The treated fragments (200 ng) and the BSMV vector (20 ng) were mixed, heated to 66°C for 2 min, and then cooled slowly to room temperature. We transformed 10 μl of the mixture into Escherichia coli using the heat shock method. Positive clones were screened using colony PCR and verified via sequencing. The positive clones were shaken, and the plasmids were extracted for follow-up experiments. The extracted viral vector plasmids were transferred into A. tumefaciens GV3101 for triticale inoculation.

A. tumefaciens carrying BSMV-phytoene desaturase BSMV::asTaPDS constructs (BSMV-PDS) induce photobleaching or yellow–orange coloration in the silenced tissue due to depletion of enzymes involved in biosynthesis of carotenoid pigments or chlorophyll, respectively (Lee et al., 2015). Thus, these constructs may be used as positive controls for gene silencing. Triticale plants were inoculated with BSMV-PDS, BSMV-γb, or BSMV-TwNAC01 for about a week (two to three leaves were treated per plant). After an additional 2 weeks of growth, white stripes began to appear on the leaves due to the expression of the indicator gene. At this point, samples of the leaves were taken and stored at −80°C. Total RNA was extracted from these samples for quantitative reverse transcription PCR (qRT-PCR). The RWC of the leaves was determined following the methods of Flexas et al. (2006), MDA content in the leaves was estimated following Chen and Wang (2002), and H₂O₂ content in the leaves was determined using an H₂O₂ measurement kit (Nanjing Jiancheng Science and Technology Co., Ltd). Stomatal conductance, net photosynthesis rate, transpiration rate, and intercellular CO₂ concentration were measured using a LI-6400 portable photosynthesis meter (Li-Cor). All these physiological parameters were determined in four replications, respectively.

Microsoft Excel was used for data analysis, and one-way ANOVA was conducted using SPSS Statistics 22.0 software to assess significance of differences. The data were analyzed using Student’s t-test and the difference was considered significant statistically at p < 0.05.

Using primers designed based on the sequence of Unigene c51971 (GSP-R/F in Supplementary Table S1), we amplified an intermediate sequence from the extracted triticale RNA (Supplementary Figure S1A) that was 502-bp long (Supplementary Figure S1B). Based on this intermediate sequence, we used 5′-RACE to amplify the 224 bp 5′ sequence (Supplementary Figure S1C), and 3′-RACE to amplify the 557 bp 3′ sequence (Supplementary Figure S1D). Splicing of the 5′ and 3′ sequences based on the intermediate sequence (Unigene c51971) yielded a fill-length ORF of 1,059 bp. Using specific primers designed out of this ORF sequence, we successfully amplified the gene (Supplementary Figure S1E). Transformation of this gene fragment into E. coli via the pMD19-T vector confirmed the expression of a 1,059-bp sequence (Supplementary Figure S1F). The predicted amino acid sequence of the gene was 352-bp long and had over 95% homology with the NAC amino acid sequences from barley, wheat, and other plants. We thus inferred that the cloned gene was a triticale NAC gene, which was designated TwNAC01. This gene has been submitted to GenBank (accession number MG736919).

The molecular formula of the encoded protein was predicted to be C₁₇₂₂H₂₆₄₂N₄₆₄O₅₂₂S₁₉, with a predicted molecular weight of 38805.86 kDa, and a theoretical isoelectric point of 5.44. The total number of positive/negative charge residues predicted in this protein were 46/37, and the atomic composition was C₁₇₂₂, H₂₆₄₂, N₄₆₄, O₅₂₂, and S₁₉. The extinction coefficient of the predicted protein was 46,996, and its absorbance at a wavelength of 280 nm was 1.211 l g⁻¹ cm⁻¹. The total average hydrophobic coefficient of the predicted protein was −0.494, indicating that the putative protein was hydrophilic.

A NJ phylogenetic tree based on sequence similarity showed that the triticale TwNAC01 protein formed a clade with NAC proteins from Aegilops tauschii (XP-020161331), and Hordeum vulgare (KAE8777325 and CBZ41151; Figure 1A). In particular, the amino acid sequence of TwNAC01 was more than 97% similar to Ae. tauschii protein AtNAC92 (XP20161331.1; Figure 1B). The predicted TwNAC01 sequence contained a conserved NAM-superfamily domain composed of 129 consecutive amino acids at the N-terminus (between amino acid 20 and 148) and three transcriptional activation domains at the C-terminus (Figure 1C).

Localization prediction analysis indicated that the TwNAC01 protein was not located in the chloroplasts or mitochondria. This protein was unlikely to be a chloroplast transport peptide, mitochondrial transport peptide, or signal peptide. The target protein was found in other organelles secretory pathways. Further study with the Protcomp analysis indicated that the predicted protein was located in the nucleus. The shear site was the 62th amino acids with the maximum value of 0.113, and the comprehensive splicing site was the 62th amino acids with the maximum value of 0.107 (No signal peptide; Table 1). The shear site was consistent with the predicted upper transmembrane region and signal peptide (Table 1). In the control group (35S:EGFP), the EGFP localization signal was dispersed throughout the cell, with the strongest signals originating primarily from the cell membrane and nucleus (Figure 2). In the treatment group (35S:TwNAC01-EGFP), the EGFP protein signal was restricted to the nucleus (Figure 2). This suggested that the fusion protein was located in the nucleus, as was predicted by our bioinformatics analysis.

The TwNAC01 gene was significantly upregulated (p < 0.01) in triticale roots and grains of drought-stressed plants as compared to the control (Figure 3A); TwNAC01 was also upregulated with respect to the control in the leaves (p < 0.05), but upregulated insignificantly with respect to the control in the stems. This suggested that the triticale TwNAC01 gene was upregulated in response to drought stress, with the strongest upregulation found in the grain, followed by the root, leaf, and stem.

After 1- and 24-h NaCl treatment, TwNAC01 expression was significantly more upregulated in the roots than in the leaves (Figure 3B). In contrast, after 24 h of cold and PEG6000-induced dehydration stress, TwNAC01 was significantly upregulated in the leaves as compared to the roots (although TwNAC01 was significantly upregulated in the roots as compared to the leaves after 12-h PEG6000 treatment; Figures 3C,D). After 24 h of MeJA and ABA stress, TwNAC01 was significantly upregulated in the roots as compared to the leaves; in response to these stressors, TwNAC01 expression levels appeared to increase over time (Figures 3E,F). After ABA treatment, TwNAC01 gene expression levels in roots were significantly higher than those in leaves throughout the time course (Figure 3F). Overall, the data indicated that the TwNAC01 gene played a role in the stress response of the triticale roots and leaves.

PCR analysis of E. coli transformed with the pCAMBIA1300-35S overexpression vector recovered a 1,059-bp band, indicating that transformation had been successful and that the target gene was ligated to the vector (Supplementary Figure S2A). After restriction endonuclease digestion, the vector skeleton (10 kb) and a band slightly longer than 1,000 bp were obtained (Supplementary Figure S2B), again indicating that the recombinant expression plasmid containing the target gene were successfully constructed. The A. tumefaciens solution containing the expression vector plasmid was also analyzed using PCR, and a 1,059-bp band was confirmed (Supplementary Figure S2C). After the plasmid was introduced into A. thaliana inflorescences using the floral dip method, three T₀ plants expressing TwNAC01 were identified via 1/2 MS Kan + medium followed by PCR verification. Derived from these T₀ lines, three T₃ lines were confirmed to express TwNAC01.

After 2 h of treatment at 25°C, while the leaves of A. thaliana WT and those expressing the VC had curled slightly, the leaves of the transgenic A. thaliana lines overexpressing TwNAC01 (TwNAC01-1, TwNAC01-2, and TwNAC01-3) exhibited no obvious curling (Figure 4A). After 5 h at 25°C, the leaves of the WT and VC plants were obviously curled, while the leaves of the TwNAC01-overexpressing lines were only slightly curled. After 8 h of dehydration, the leaves of the WT and VC plants were noticeably withered and crumpled in appearance; although the leaves of the transgenic lines were also somewhat withered, the observed degree of dehydration was much less severe (Figure 4A).

However, the three transgenics slowed down water loss (29.90%–49.30%) significantly, especially during the first 2 h. As drying continued, water loss rates increased almost linearly in all lines (Figure 4B). The overall data indicated that water loss rates in the transgenic lines were significantly (p < 0.05) lower (16.77%–58.77%) than those in the WT (31.02%–68.18%) and VC (27.07%–62.86%) lines (Figure 4B). This result should attribute to the longer roots (9.90 ± 0.03–10.20 ± 0.24 cm) in transgenic lines relative to the WT (6.52 ± 0.41 cm) and VC (8.18 ± 0.17 cm) counterparts (differences of 1.5-fold and 1.2-fold, respectively; Figures 5A,B).

Although the relative leaf water contents (62.03%–64.70%) of the three transgenic Arabidopsis lines were slightly higher than those of VC (55.92%) and WT (48.09%) lines, these differences were not significant (Figure 6A). However, leaf electrical conductivity (reflecting electrolyte leakage and thus membrane damage) was significantly greater in the VC (63.67%) and WT (71.60%) lines as compared to the transgenic lines (32.07%–32.60%; Figure 6B). Hydrogen peroxide (18.29 μmol g⁻¹ FW) and MDA (6.27 μmol g⁻¹ FW) levels were significantly greater in the leaves of the WT line as compared to all other lines; there were no significant differences in H₂O₂ or MDA levels between the VC plants (14.37 μmol g⁻¹ FW and 5.81 μmol g⁻¹ FW) and any of the transgenic lines (11.85–12.12 μmol g⁻¹ FW and 2.32–2.66 μmol g⁻¹ FW; Figures 6C,D).

After drought stress, TwNAC01 was upregulated in the roots and leaves of the transgenic plants as compared to the VC and WT plants; in all three transgenic lines, TwNAC01 gene expression was significantly greater in the roots than it in the leaves (Figure 6E). On average, TwNAC01 gene expression levels in the transgenic A. thaliana lines were 8-fold and 38-fold greater than in the VC and WT plants, respectively. Thus, in response to drought stress, TwNAC01 was upregulated in transgenic A. thaliana overexpressing TwNAC01 as compared to mock-transformed and WT controls.

A 327-bp sequence was amplified from the triticale cDNA as mentioned above using the virus-induced gene silencing (VIGS) primers (Supplementary Figure S3A). After transformation of the amplified sequence into E. coli, positive clones were identified via PCR amplification (Supplementary Figures S3B,C). After drought stress, TwNAC01 gene expression levels in the control were significantly greater than those in the TwNAC01-silenced plants (BSMV-TwNAC01; Figure 7). In contrast, drought stress significantly upregulated TwNAC01 in the empty vector (BSMV-γb) and indicator-gene (BSMV-PDS) plants as compared to the control. This indicated that drought stress upregulated the triticale TwNAC01 gene; TwNAC01 was downregulated significantly even under drought conditions when it was silenced (Figure 7).

About 2 weeks after inoculation with the BSMV vectors, the indicator-gene (BSMV-PDS) plants began to exhibit symptoms of stripe mosaic virus. On the 20th day after inoculation, large areas of the leaves of the BSMV-PDS plants were bleached, while the leaves of empty vector (BSMV-γb) and TwNAC01-silenced (BSMV-TwNAC01) plants showed slight bleaching (Figure 8A). In general, the growth potential of each of the inoculated groups (BSMV-γb, BSMV-PDS, and BSMV-TwNAC01) was weaker than that of the control (Figures 8B,C). The mean RWC (57.64%) of the leaves of BSMV-TwNAC01 was significantly lower than that of the leaves of the control (71.63%; Figure 8D). The roots of the TwNAC01-silenced plants were significantly shorter (3.95 cm) than those of the control (5.58 cm), BSMV-γb (5.04 cm), and indicator-gene plants (4.95 cm; Figures 8E,F). The results indicated that TwNAC01 gene silencing inhibits triticale root development and significantly reduced triticale growth.

After drought stress, levels of H₂O₂ (54.17 μmol g⁻¹ FW) and MDA (41.49 μmol g⁻¹ FW) in the leaves of BSMV-TwNAC01 plants were significantly higher (Figures 9A,B), while RWC was significantly lower (57.64%; Figure 9C) than those in the leaves of uninfected control plants (65.51%–71.63%). This result demonstrated that the triticale leaves were more stressed by drought when the TwNAC01 gene was silenced, i.e., the stress-resistant ability of the plant decreased after silencing the TwNAC01 gene. The data suggested that TwNAC01 played an important role in coping with stress. Furthermore, net photosynthetic rate, stomatal conductance to H₂O, intracellular CO₂ level, and transpiration rate were significantly lower in the BSMV-TwNAC01 leaves as compared to the control (Figures 9D–G). Net photosynthetic rate and intracellular CO₂ level were significantly lower than the control in the BSMV-γb and BSMV-PDS leaves (Figures 9D,F), while transpiration rate was significantly lower than the control in the BSMV-PDS leaves (Figure 9G).