Identi cation and characterization of a novel NAC transcription factor gene from triticale (x Triticosecale Wittmack)


 The NAM, ATAF, and CUC (NAC) family of transcription factors plays several important roles in plants, helping to regulate plant growth, development, senescence, and the response to biotic and abiotic stressors. NAC proteins also act as molecular switches, modulating hormonal responses to stress.A novel coding sequence (1059 bp) was cloned from hexaploid triticale. The putative protein encoded by this sequence (352 amino acids) was more than 95% similar to the amino acid sequence of a NAC protein from Aegilopsis tauschii (goatgrass; XP020161331), and phylogenetic analysis indicated that the novel gene formed a clade with goatgrass, Triticum turgidum , and barley. The novel protein contained a conserved nature actomyosin (NAM) domain (129 consecutive amino acids) between the 20 th and 148 th amino acids at the N-terminus and three transcriptional activation regions at the C-terminus. TwNAC01 was localized to the nucleus. Based on this evidence, the novel gene was identified as a triticale NAC gene and designated TwNAC01 (GenBank accession no. MG736919). After exposure to drought, Macrogol 6000 (PEG6000), NaCl, cold, methyl jasmonate (MeJA), and abscisic acid (ABA), TwNAC01 expression levels were greatest in triticale roots, followed by leaves and stems. Transgenic Arabidopsis thaliana overexpressing TwNAC01 had significantly lower leaf water loss rates and significantly longer roots than wild-type A. thaliana. Virus-induced silencing of the TwNAC01 gene in triticale delayed root development and decreased taproot length. Under drought stress, leaves of TwNAC01- silenced triticale had higher levels of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) than the leaves of the wild type (WT), as well as lower relative water content (RWC), net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, and transpiration rate.Gene overexpression and silencing experiments suggested that TwNAC01 improves plant stress tolerance by increasing taproot length, regulating the water content of the plant leaves, reducing MAD and H 2 O 2 content, and adjusting respiration rate.


Introduction
Triticale (⋅ Triticosecale Wittmack) is a new allopolyploid crop derived from the intergeneric hybridization of wheat (Triticum) and rye (Secale), followed by chromosome doubling (Zilinsky et al., 1974). This crop, which can be used as both a food crop and a forage crop, combines the high grain yield and good quality of wheat with the strong stress resistance of rye (Cao et al., 2011;Sun et al., 2002). Because triticale exhibits strong stress resistance in cultivation, the mining of its resistance genes is important for the molecular breeding of even more stress-resistant triticale varieties.
During growth and development, plants are often affected by a variety of natural adverse environmental factors, such as high temperatures, drought, salinity, and extreme weather. To survive despite these challenges, plants use series of defense mechanisms to resist and tolerate a variety of biotic and abiotic stresses (Cramer et al., 2010;Pinheiro et al., 2011). By binding to cis-acting elements in the target gene promoter, transcription factors act as molecular switches for gene expression, activate or inhibit the expression of target genes, regulate the expression of plant-related genes, and participate in the stress response (Puranik et al., 2012;Nakashima et al., 2012). Many transcription factor families are found in plants, including NAC, WRKY, DREB, and MYB transcription factors (Puranik et al., 2012). Of these, the plantspeci c NAC transcription factor family has the most members (Kim et al., 2014;Perez-rodriguez et al., Shihezi University (Xinjiang, China). To eliminate the effects of natural rainfall, we build a rain shelter over the drought-stress plot before the owering stage. Plants owering at the same time were selected during the owering stage. The control area was irrigated normally. In the drought stress area, irrigation was stopped after the triticale plants reached the heading stage. Both control and experimental plants were visually assessed daily for signs of drought stress. Proline, MDA, electrical conductivity, and chlorophyll levels in the leaves of both sets of plants were also assessed daily as described previously (Chen et al., 2002). During drought stress, proline and MDA levels increased signi cantly, while soil moisture content levels decreased signi cantly ( Figure S1). When physical indicators of drought stress were observed, the roots, stems, ag leaves, and young grains of both the experimental and control triticale plants were collected.
Full-length 5'-and 3'-RACE RNA was extracted from the triticale leaves using Hipure HP Plant RNA Mini Kits (Magen). With these RNA sequences as templates, we performed reverse transcriptase PCR (RT-PCR) to synthesize cDNA sequences using SMARTScribe genome Reverse Transcriptase (TaKaRa, China). Synthesized cDNA was stored at − 20°C until use. Based on the RNA sequence of Unigene c51971 (708 bp), which was obtained via RNA-Seq sequencing, we designed speci c primers for 3'-and 5'-RACE using SMARTer RACE kits (Clonetech; Schedule 1). The RACE procedure was as follows: 94°C for 2 min, 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, 72°C for 35 min, and 16°C for 10 min. The PCR products were recovered and puri ed using 1.0% agarose gel electrophoresis. The puri ed PCR products were ligated to the pMD19-T vector (TaKaRa, China) and transformed into TOPO10 cells (TIANGEN, China). Positive clones were identi ed and sequenced. The open reading frame (ORF) of the full-length cDNA sequence was obtained by splicing the sequencing results using an on-line tool (CAP3;http://doua.prabi.fr/software/cap3) (Shang et al., 2018) and National Center for Biotechnology Information Basic Local Alignment Search Tool (NCBI-BLAST). Sequence alignment analysis preliminarily identi ed the sequence as a triticale NAC gene and predicted the positions of the start and stop codons. We then designed a primer pair to amplify the full-length gene (Schedule 1); this gene was preliminarily designated TwNAC01.

Analysis of the triticale TwNAC01 sequence
We used the NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/or nder/) to identify sequences homologous to the ORF and coding sequences of TwNAC01. We identi ed conserved structures in the TwNAC01 gene using Smart (http://smart.embl-heidelberg.de/). We analyzed the physical and chemical properties of the predicted TwNAC01 protein, as well as its hydrophobicity, using the ExPASy server (https://web.expasy.org).
Multi-alignment of TwNAC01 and other NAC proteins in different species was conducted on DNAMAN. We predicted the subcellular location of the protein using Protcomp and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP). The relevant sequences were aligned using MEGA (version 10.0) (Shang et al., 2018) and MegAlign (DNAStar).

Real-time uorescence quantitative PCR (qPCR)
The plants to be used for qRT-PCR were cultured at 25°C with a normal watering regime, under a 12 h light/12 h dark cycle in an arti cial climate box. When the seedlings had two leaves and one main shoot, they were transplanted in hydroponic boxes. Three hydroponic boxes were allocated to each treatment; each box had 12 holes, and each hole had 5 seedlings. 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 one of the following: 1 L of 20% PEG6000, 200 mM NaCl, 100 µM MeJA, 100 µM ABA. 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. After collection, the materials were frozen in liquid nitrogen and then transferred to a freezer at − 80°C.
RNA was extracted from the triticale materials collected from each of the six groups [cold (4°C), drought, 20% PEG6000, 200 mM NaCl, 100 µM MeJA, and 100 µM ABA using Hipure HP Plant RNA Mini Kits (Magen, China), and cDNA was synthesized using 5XAll-ln-One RT MasterMix (abm, Canada) with speci c primers (RT-PCR primer pair; Schedule 1). The wheat actin gene was used as internal reference for real-time qPCR (Schedule 1). 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 su cient ddH 2 O to make 10 µL. qPCR ampli cations 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 ampli cation 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 et al., 2001).

Sub-cellular localization of the TwNAC01 protein
The coding sequence of the TwNAC01 gene was cloned into the plant subcellular expression vector pCAMBIA1301S Enhanced Green Fluorescent Protein (EGFP; GenBank accession no. E17099). Insertion primers containing BamHI-XbaI restriction sites (Schedule 1) were designed and ligated using a ClonExpressII one-step cloning kit (TaKaRa, Dalian, China). After veri cation via sequencing, the recombinant plasmid and the empty pCAMBIA1301S (EGFP) vector were transformed into Agrobacterium tumefaciens GV3101 (TaKaRa, China) (Mao et al., 2014). A. tumefaciens carrying the recombinant plasmid or the empty vector (TaKaRa, China) were cultured on Luria-Bertani (LB) medium containing Kan + and Rif+. When the OD600 of the bacterial solution was 0.5-0.6, the bacterial solution was collected and resuspended in infection buffer [10 mM MgCl 2 , 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 an arti cial climate box 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 cultured in darkness for 36 hours. 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) uorescence signals in the tobacco leaves were observed using a Fluo-View confocal microscope (FV300; Olympus, Japan).

Generation of transgenic Arabidopsis overexpressing TwNAC01
To obtain transgenic Arabidopsis plants, the coding sequence of TwNAC01 containing the termination codon was ampli ed by RT-PCR and cloned into the KpnI and XbaI restriction sites of the pCAMBIA1300-35S vector (Clontech, TaKaRa, China) under the control of the 35S promoter of the cauli ower mosaic virus (CMV). The primers containing the KpnI and XbaI restriction sites are listed in Additional le 2: Table S1. The recombinant vector pCAMBIA1300-35S-TwNAC01 and the empty vector pCAMBIA1300-35S-VC were introduced into A. tumefaciens strain GV3101 (TaKaRa, China). Finally, transgenic Arabidopsis plants were generated using the A. tumefaciens-mediated oral dipping method (Clough and Bent et al., 1998). To generate homozygous progeny, T1 and T2 seeds were selected on kanamycin (50 mg/L) plates. T3 transgenic A. thaliana and wild-type A. thaliana plants were watered once at the rosette stage and then subjected to drought stress for 25 days. After 25 days of drought stress, RNA was extracted from the leaves and roots of both transgenic and wild-type A. thaliana using Hipure HP Plant RNA Mini Kits (Magen, China). TwNAC01 gene expression levels were then measured using semi-quantitative analysis with gene-speci c primers [Table S1; please see the section "Real-time uorescence quantitative PCR (qPCR)" for details].
Representative lines overexpressing TwNAC01 were used for further analysis.

Drought resistance of transgenic A. thaliana overexpressing TwNAC01
We then measured various stress-related physiological indexes in 35-day-old transgenic A. thaliana overexpressing TwNAC01, mock transformed A. thaliana (transformed with the empty vector), and wild-type A. thaliana. Leaf relative water content (RWC) was determined following the methods of Flexas et al. (2006), leaf electrical conductivity was determined following Chen Aikui et al. (2010), leaf MDA content was determined following Chen and Wang et al. (2002), and leaf H 2 O 2 content was determined using an H 2 O 2 measurement kit (China Nanjing Jiancheng Science and Technology Co., Ltd). We also determined the rate of water loss in the leaves. Five rosette leaves from each group of A. thaliana plants (wild-type, empty vector, and the three TwNAC01-overexpression lines) were collected, transferred to lter paper, and placed in a constant temperature incubator at 25°C. Leaves were weighed every hour for 8 h and photographed after 2 h, 5 h, and 8 h. Water loss was judged based weight loss and on 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.
Speci c primers were designed for the PCR ampli cation of silencing fragments based on the 3'-UTR region of TwNAC01 gene (Schedule 1), 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 treated with T4 DNA polymerase, and the BSMV-rb vectors were digested with the ApaI restriction enzyme. A nal concentration of 5 mM deoxythymidine triphosphate (dTTP) was added to the carrier reaction system, and the system was allowed 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 T4 DNA polymerase. The treated fragments (200 ng) and the carrier (20 ng) were mixed, heated to 66°C for 2 min, and then cooled slowly to room temperature. We transformed 10 µl of the mixed product into Escherichia coli using the heat shock method. Positive clones were screened using colony PCR and veri ed 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.
Stress resistance of triticale after TwNAC01 gene silencing.
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, BSMVγb, or BSMV-TwNAC01 for about a week (two to three leaves were treated per plant). After an additional two weeks of growth, white stripes began to appear on the leaves due to the action 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 determined following Chen et al. (Chen et al., 2002), and H 2 O 2 content in the leaves was determined using an H 2 O 2 measurement kit (Nanjing Jiancheng Science and Technology Co., Ltd). Stomatal conductance, net photosynthesis rate, transpiration rate, and intercellular CO 2 concentration were measured using a LI-6400 portable photosynthesis meter (Licor).

Statistical analysis
Microsoft Excel was used for data analysis and Origin and One-way ANOVA was conducted on SPSS Statistics 22.0 software to assess the signi cant differences. The data were analyzed using Student's t tests; we considered P < 0.05 statistically signi cant.

Result
The full-length TwNAC01 gene Using primers designed based on the sequence of Unigene c51971 (GSP-R/F; Schedule 1), we ampli ed an intermediate sequence from the extracted triticale RNA ( Figure S2a) that was 502 bp long ( Figure S2b). Based on this intermediate sequence, we used 5'-RACE to amplify the 224 bp 5' sequence ( Figure S2c) and 3'-RACE to amplify the 557 bp 3' sequence ( Figure S2d). Splicing of the 3' and 5' sequences based on the intermediate sequence (Unigene c51971), yielded a ll-length ORF of 1059 bp. Using speci c primers designed based on this sequence, we successfully ampli ed the gene ( Figure S2e). Transformation of this gene fragment into E. coli via the pMD19-T vector con rmed the expression of a 1059 bp sequence ( Figure  S2f). The predicted amino acid sequence of the gene was 352 bp long and had more than 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 we designated TwNAC01. This gene has been submitted to GenBank (accession number MG736919).

Gene sequence analysis
A NJ phylogenetic tree based on these highly similar sequences showed that the triticale TwNAC01 protein formed a clade with NAC proteins from Aegilops (XP-020161331), Triticum houdeum (KAE8777325), and Hordeum vulgare (CBZ41151) (Fig. 1a). In particular, the amino acid sequence of TwNAC01 was more than 95% similar to the wheat (T. aestivum) protein TaNAC20 (KY461026.1) and the Aegilops tauschii protein AtNAC92 (XM020305742.2). The predicted TwNAC01 protein sequence was highly homologous to NAC sequences from barley, wheat, goatgrass, and durum wheat. Sequence alignment revealed that TwNAC01 shared 97.3% and 83.8% similarity with that of XP020161331 and KAE87777325,TwNAC01 also belonged to the NAM subgroup (Fig. 1b) .The predicted TwNAC01 sequence contained a conserved NAM-superfamily domain composed of 129 consecutive amino acids at the N-terminus (between ammino acid 20 and 148) and three transcriptional activation domains at the C-terminus (Fig. 1c).

Subcellular localization in the nucleus
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 the "_",( in other organelles) secretory pathway. These results, in conjunction with the Protcomp analysis (see section "Analysis of the triticale TwNAC01 sequence"), indicated that predicted protein was located in the nucleus. The shear site was consistent with the predicted upper transmembrane region and signal peptide (Table 1) That is, the shear site was 62 amino acids long with a maximum of 0.113, and the comprehensive splicing site was 62 amino acids with a maximum value of 0.107 for the signal peptide to show: NO (Table 1). In the control group, the EGFP localization signal was dispersed throughout the cell, with the strongest signals originating primarily from the cell membrane and nucleus (Fig. 2). In the treatment group, the EGFP protein signal was restricted to the nucleus (Fig. 2). This suggested that the fusion protein was located in the nucleus, as was predicted by our bioinformatics analysis.

Analysis of TwNAC01 gene expression in triticale under stress
The TwNAC01 gene was signi cantly upregulated (P < 0.05) in triticale roots and grains after drought treatment as compared to the control; TwNAC01 was also upregulated with respect to the control in the stem and leaf tissues, but these differences were not signi cant (Fig. 3a). 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 24 h of salinity, MejA, and ABA stress, TwNAC01 was signi cantly upregulated in the roots as compared to the leaves; in response to these stressors, TwNAC01 expression levels appeared to increase over time (Fig. 3e, 3f). After 1 h and 24 h of NaCl treatment, TwNAC01 expression was signi cantly more upregulated in the roots than in the leaves (Fig. 3b). In contrast, after 24 h of cold stress and PEG6000induced dehydration, TwNAC01 was signi cantly upregulated in the leaves as compared to the roots (although TwNAC01 was signi cantly upregulated in the roots as compared to the leaves after 12 h of PEG6000 treatment; Fig. 3d, 3c). This might suggest that the strength of the cold and drought response in the roots increased over time, and that the leaves are more sensitive to cold than the roots. After ABA treatment, TwNAC01 gene expression levels in roots were consistently signi cantly higher than those in leaves at the same time point (Fig. 3f). Thus, it was likely that the TwNAC01 gene played a role in the stress response of the triticale roots and leaves.

Con rmation of transgenic A. thaliana lines overexpressing TwNAC01
PCR analysis of E. coli transformed with the pCAMBIA1300-35s overexpression vector recovered 1059 bp band, indicating that transformation had been successful and that the target gene was connected to the vector ( Figure S3a). After restriction endonuclease digestion, the vector skeleton (10 kb) and a band slightly longer than 1000 bp were obtained ( Figure S3b), indicating that the recombinant expression plasmid containing the target gene had been successfully constructed. The A. tumefaciens solution containing the expression vector plasmid was also analyzed using PCR, and a 1059 bp band was recovered ( Figure S3c). This indicated that the recombinant plasmid had been successfully introduced into A. tumefaciens. After the plasmid was introduced into A. thaliana in orescences using the oral dip method, three T 0 plants expressing TwNAC01 were identi ed via PCR and 1/2 MS Kan + medium. From these T 0 lines, three T 3 lines were generated that were con rmed to express TwNAC01.

Root length and leaf water loss rate in transgenic A. thaliana overexpressing TwNAC01
After two hours of drying at 25°C, the leaves of wild-type A. thaliana and those of A. thaliana expressing the empty vector had curled slightly, while the leaves of the transgenic A. thaliana lines overexpressing TwNAC01 (TwNAC01-1, TwNAC01-2, and TwNAC01-3) exhibited no obvious curling (Fig. 4a). After ve hours of drying at 25°C, the leaves of the wild-type and empty-vector plants were obviously curled, while the leaves of the TwNAC01-overexpression lines were only slightly curled. After eight hours of dehydration, the leaves of the wild-type and empty-vector 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 (Fig. 4a). Water loss rate in the wild-type, empty-vector, and transgenic plants increased signi cantly over time (P < 0.05) (Fig. 4b). After one hour of drying, water loss rates were similar across the three lines, and, as drying continued, water loss rates increased linearly in all lines (Fig. 4b). However, for most of the drying time, water loss rates in the transgenic lines were signi cantly (P < 0.05) lower than those in the wild-type and empty-vector lines (Fig. 5b). The three transgenic lines had signi cantly longer roots than the wild-type and empty-vector lines (differences of 1.5-fold and 1.2-fold, respectively; Fig. 5a, 5b).

Physiological indexes of stress resistance in A. thaliana overexpressing TwNAC01
Although the relative leaf water contents of the three transgenic Arabidopsis lines were slightly higher than those of empty-vector and wild-type lines, these differences were not signi cant (Fig. 6a). However, leaf electrical conductivity (re ecting electrolyte leakage and thus membrane damage) was signi cantly greater in the empty-vector and wild-type lines as compared to the transgenic lines (Fig. 6b). H 2 O 2 and MDA levels were signi cantly greater in the leaves of the wild-type line as compared to all other lines; there were no signi cant differences in hydrogen peroxide or MDA levels between the empty-vector plants and any of the transgenic lines (Fig. 6c, 6d).
After drought stress, TwNAC01 was upregulated in the roots and leaves of the transgenic plants as compared to the empty-vector and wild-type plants; in all three transgenic lines, TwNAC01 gene expression was signi cantly greater in the roots than the leaves (Fig. 6e). On average, TwNAC01 gene expression levels in the transgenic A. thaliana lines were 8-fold and 38-fold greater than the empty-vector and wild-type plants, respectively. Thus, in response to drought stress, TwNAC01 was upregulated in transgenic A. thaliana overexpressing TwNAC01 as compared to mock-transformed and wild-type A. thaliana.

Expression of TWNAC01 after VIGS in triticale under drought stress
A 327 bp sequence was ampli ed using the VIGS primers ( Figure S4a, Fig. 5). After transformation of the ampli ed sequence into E. coli, positive clones were identi ed via PCR ampli cation ( Figure S4b, S4c). After drought stress, TwNAC01 gene expression levels in the control plants were signi cantly greater than those in the TwNAC01-silenced plants (BSMV-TwNAC01). In contrast, drought stress signi cantly upregulated TwNAC01 in the empty-vector (BSMV-γb) and indicator-gene (BSMV-PDS) plants as compared to the control plant. This indicated that drought stress upregulated the triticale TwNAC01 gene; after TwNAC01 gene silencing, TwNAC01 was downregulated, even under drought conditions (Fig. 7).

Phenotypic implications of TwNAC01 gene silencing in triticale
About two weeks after inoculation with the BSMV vectors, the indicator-gene (BSMV-PDS) plants began to exhibit symptoms of stripe mosaic virus. At 20 days after inoculation, large areas of the leaves of these plants were bleached, while the leaves of empty-vector (BSMV-γb) and TwNAC01-silenced (BSMV-TwNAC01) plants showed slight bleaching (Fig. 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 group (Fig. 8b, 8c). The mean RWC of the leaves of BSMV-TwNAC01 was signi cantly lower than that of the leaves of the CK (Fig. 8d). The roots of the TwNAC01-silenced plants were signi cantly shorter than those of the control, empty-vector, and indicator-gene plants (Fig. 8e, 8f). This suggested that the TwNAC01 gene substantially affects triticale root growth, and that TwNAC01 gene silencing inhibits triticale root development. This showed that silencing the TwNAC01 gene signi cantly reduced triticale growth.

Physiological indexes of drought stress and photosynthesis in TwNAC01-silenced triticale
After drought stress, levels of H2O2 and MDA in the leaves of BSMV-TwNAC01 plants were signi cantly higher than those in the leaves of uninfected control plants (Fig. 9a, 9c), while RWC was signi cantly lower (Fig. 9b). This demonstrated that the triticale leaves were more stressed by drought when the TwNAC01 gene was silenced. That is, the stress-resistance ability of the plant decreased after TwNAC01 gene silencing. This suggested that TwNAC01 played an important role in triticale stress resistance.Net photosynthetic rate, stomatal conductance to H 2 O, intracellular CO 2 level, and transpiration rate were signi cantly lower in the BSMV-TwNAC01 leaves as compared to the control ( Fig. 9d-g); net photosynthetic rate and intracellular CO2 level were signi cantly lower than the control in the BSMV-γb and BSMV-PDS leaves (Fig. 9d, 9f), while transpiration rate was signi cantly lower than the control in the BSMV-PDS leaves (Fig. 9g). Net photosynthetic rate (Fig. 9d), stomatal conductance (Fig. 9e), intercellular CO 2 concentration ( Fig. 9f), and transpiration rate (Fig. 9g) of control, BSMV-γb, BSMV-PDS, and BSMV-TwNAC01 triticale leaves decreased gradually as drought stress increased. Thus, TwNAC01 gene silencing signi cantly affected photosynthesis-related indexes in the leaves of BSMV-infected plants subjected to drought stress, again demonstrating that TwNAC01 silencing weakened the stress resistance of triticale.

Discussion
NAC transcription factors are considered the most important family of transcription factors in plants; these transcription factors play various important roles in the stress response, as well as in the regulation of plant growth and development (Xu et al., 2015). Stress-related NAC transcription factors have been well examined in wheat, rice, and Leymus triticoides, but relatively few studies have explored stress-related NAC transcription factors in allohexaploid triticale. Here, we used RACE RNA-seq and RT-PCR to clone the rst putative NAC gene from heterohexaploid triticale. This putative NAC gene had more than 95% similarity with known NAC genes from other crops, including common wheat, goatgrass, and durum wheat. The molecular formula of the encoded protein was predicted to be C 1722 H 2642 N 464 O 522 S 19 , with a predicted molecular weight of 38805.86 M r , 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 C 1722 , H 2642 , N 464 , O 522 , and S 19 , The extinction coe cient of the predicted protein was 46,996, and its absorbance at a wavelength of 280 nm was 1.211 L/(g·cm). The total average hydrophobic coe cient of the predicted protein was − 0.494, indicating that the putative protein was hydrophilic. These ndings were consistent with our phylogenetic analysis. In addition, the predicted protein sequence of the putative NAC gene included a conserved NAM domain between the 20th and 148th amino acids at the N-terminus, as well as three transcriptional activation regions at the C-terminus. We thus concluded that the putative gene was indeed an NAC gene. This gene was designated TwNAC01.
Our results showed that TwNAC01 was upregulated by a variety of abiotic stressors and signal molecules, including salinity, drought, PEG6000, and ABA. Several studies have shown that about 20-25% of plant NAC genes respond to stress treatments and participate in stress alleviation (Puranik et al., 2012;Nuruzzaman et al., 2010;Fang et al., 2008). In addition, A. thaliana overexpressing the wheat NAC genes TaNAC2 and TaNAC67 exhibited signi cantly improved tolerance of drought, salinity, and cold as compared to the wild-type (Mao et al., 2014). Similarly, the overexpression of wheat NAC genes (TaNAC2a, TANAC4a, TaNAC6, TaNAC7, TaNAC13, and TaNTL5) in tobacco signi cantly improved drought tolerance (Tang et al., 2012). Previous studies have also shown that NAC gene expression increases in response to drought stress (Wu et al., 2009;Chen et al., 2014;Nakashima et al., 2007). Consistent with this, TwNAC01 expression was signi cantly upregulated in the drought-stressed plant roots and young grains as compared to the unstressed controls. This suggested that TwNAC01 expression is induced by drought stress, and that this gene plays an important role in the response to drought in triticale.NAC genes have also been shown to be upregulated in aging plant tissues, by treatment with plant signal molecules (e.g., ABA, ethephon, JA, and SA) (Jensen et al., 2010;Tang et al., 2012;Bu et al., 2008;Xia et al., 2010), and by exposure to ethylene and methyl jasmonate (Scharrenberg et al., 2003). Here, the upregulation of TwNAC01 in response to ABA and various abiotic stressors indicated that the abiotic stress process may be regulated by the ABA hormone in triticale.The upregulation of TwNAC01 in response to stress was stronger in the roots as compared to the leaves, consistent with a previous study, which showed that TaNAC4 was more strongly upregulated in wheat roots as compared to leaves and stems (Xian et al., 2010). This suggested that TwNAC01 will be overexpressed in triticale roots rst in response to stress, in order to promote the development of plant roots and reduce the damage caused by adverse conditions.
The mechanisms by which NAC transcription factors alleviate drought stress in plants have been well studied. For example, the overexpression of the NAC gene ATAF1 in A. thaliana decreased transpiration rate and increased drought tolerance . Similarly, rice overexpressing OsNAP had lower water loss rates during vegetative growth, increased sensitivity to exogenous ABA, and improved tolerance of salt, drought, and low temperature stress (Negi et al., 2018). Also in rice, NAC transcription factors regulate the expression of OsSRO1c genes, which are primarily expressed in guard cells; the overexpression of OsSRO1c increases H 2 O 2 accumulation in guard cells and reduces the number of completely open stomata, thus reducing water loss via transpiration (You et al., 2013). Finally, the SNAC3 gene in rice targets a ROS-scavenging gene, and SNAC3 overexpression upregulates this target gene (Fang et al., 2015). These previous results suggest that, when plants are under stress, the NAC gene can reduce transpiration rate by promoting plant root elongation, improving H 2 O 2 and MAD accumulation, and increasing leaf water content.
Ultimately, these factors enhance plant adaptability to adversity. Here, TwNAC01 expression was successfully silenced in triticale BSMV-TwNAC01 plants: TwNAC01 expression levels in the triticale BSMV-TwNAC01 plants were signi cantly lower than those in the control, BSMV-γb, and BSMV-PDS plants. In general, the growth potential of the TwNAC01-silenced plants was signi cantly lower than that of the other lines. In particular, the TwNAC01-silenced plants had shorter roots and reduced water content as compared to the other plants. Under drought conditions, markers of physiological stress (i.e., MDA and H 2 O 2 levels) were signi cantly increased in TwNAC01-silenced plants as compared to the controls, while relative water content and markers of photosynthetic activity (net photosynthetic rate, stomatal conductance, transpiration rate, and intercellular CO 2 concentration) were signi cantly reduced. The observed changes in these physiological indexes suggested that triticale growth and stress resistance were substantially impaired by TwNAC01 silencing.
In Conclusion, The overexpression of the triticale TwNAC01 gene in A. thaliana improved the drought resistance of A. thaliana by increasing the water retention capacity of the leaves, reducing cellular membrane damage, decreasing the production of ROS in the leaves, and promoting root elongation. In TwNAC01-silenced triticale, leaf relative water content and the root length were signi cantly decreased as compared to the control, while leaf H 2 O 2 and MDA levels were signi cantly increased. Leaf net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, and transpiration rate were also signi cantly lower in the TwNAC01-silenced plants as compared to the control. These results indicated that TwNAC01 silencing decreased the drought resistance of triticale, suggesting that the TwNAC01 gene plays an important role in the response of triticale to drought stress.

Declaration of competing interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Author Contributions
GK planned and designed the research and wrote the main manuscript text; MW performed most of the experiments and data acquisition; LR, XW, HG, SY and YL helped with experiments and data analysis; MW participated in gure preparation and manuscript organization. All the authors agreed on the contents of the paper and declare no con icts of interest.      Relative TwNAC01 expression patterns in triticale after TwNAC01 gene silencing, as determined using qRT-PCR and the 2−ΔΔCT method. Transcript levels were normalized to TaActin. CK, vector control; BSMV-γb, empty vector; BSMV-PDS, BSMV plus indicator gene; BSMV-TwNAC01, vector carrying silenced TwNAC01.
Values shown are the means ± SE of three replicates of three independent samples. Asterisks indicate statistically signi cant differences from the CK (vector control) plants (* P < 0.05; ** P < 0.01).  Physiological indexes of drought stress and photosynthesis in triticale leaves after virus-induced gene silencing using BSMV. a. H2O2 concentration. b. Relative water content (RWC