Introduction
Drought stress is one of the most important factors reducing crop yields, and affecting growth and development. This stress can simultaneously affect many traits through agronomic, morphological, physiological, biochemical and metabolic changes which occur in all plant tissues and ultimately reduce yield performance (Cochard et al., Reference Cochard, Coll, Le Roux and Améglio2002). The studies on wheat (Triticum aestivum L.) drought response mechanisms are needed to develop wheat varieties that can tolerate drought stress. Wild progenitors of common wheat are a potential source of tolerance to biotic and abiotic stresses, especially tolerance to drought stress (Budak et al., Reference Budak, Kantar and Yucebilgili Kurtoglu2013). Triticum boeoticum (2n = 2x = 14, AA) is a close relative of the A genome donor of wheat which includes suitable variability for many important traits resistance to different kinds of environmental stresses (Singh et al., Reference Singh, Ghai, Garg, Chhuneja, Kaur, Schnurbusch, Keller and Dhaliwal2007). The natural habitat of T. boeoticum is located in the northern and eastern parts of the Fertile Crescent (Dvorak et al., Reference Dvorak, Luo and Yang1998). Iran, located in the east of the Fertile Crescent, is considered as one of the main centres for the distribution of wild wheat, including T. boeoticum (Tabatabaei and Maassoumi, Reference Tabatabaei and Maassoumi2001). The origins of wild wheat, especially T. boeoticum in the northwest and west regions of Iran (east of the Fertile Crescent), have high levels of genetic diversity, and includes suitable genes to be transferred into cultivated wheat (Pour-Aboughadareh et al., Reference Pour-Aboughadareh, Mahmoudi, Moghaddam, Ahmadi, Mehrabi and Alavikia2017). T. boeoticum is more tolerant to drought than other wheat relatives, such as T. dicoccoides, T. araraticum and common wheat cultivars (Sultan et al., Reference Sultan, Hui, Yang and Xian2012). Since breeding wheat drought stress tolerance requires a remarkable level of heritable variation among wheat genotypes or their wild relatives (Ashraf, Reference Ashraf2010), genetic diversity of many plant species has so far been evaluated using different morpho-physiological characters and molecular markers.
NAM/ATAF/CUC (NAC) contains one of the greatest transcription factors or TFs families, including 149 and 106 predicted members in rice and Arabidopsis genomes, respectively. All members of this family have a variable C-terminal transcriptional regulation domain and a highly conserved N-terminal NAC domain (Ernst et al., Reference Ernst, Olsen, Skriver, Larsen and Leggio2004). NAC TFs are large gene families, which are involved in developmental processes, tension responses and plant growth regulation (Tang et al., Reference Tang, Liu, Gao, Zhang, Zhao, Zhao, Zhang and Chen2012). NAC proteins have been widespread in land plants, but no homolog has been recognized thus far in other eukaryotes (Riechmann et al., Reference Riechmann, Heard, Martin, Reuber, Jiang, Keddie, Adam, Pineda, Ratcliffe and Samaha2000). Furthermore, ATAF, a subfamily of NAC TFs, are significant adjusters of plants replies towards wounding and pathogen attack (Collinge and Boller, Reference Collinge and Boller2001). Recently, it is specified that NAC TFs have main functions in secondary wall formation (Yamaguchi et al., Reference Yamaguchi, Mitsuda, Ohtani, Ohme-Takagi, Kato and Demura2011).
Although many studies are available about the important duty of TFs in response to abiotic and biotic stresses in populus (Wang et al., Reference Wang, Zhao, Gao and Yang2017) and tobacco (Tang et al., Reference Tang, Liu, Gao, Zhang, Zhao, Zhao, Zhang and Chen2012), no report exists on the TFs in T. boeoticum wild wheat. Therefore, in order to study the effect of drought stress on the wild diploid wheat, and to understand its adaptive mechanisms at the molecular level, 10 accessions of T. boeoticum were evaluated under non-stress and drought stress conditions.
Materials and methods
Plant materials, growth conditions and stress treatment
The materials used in the greenhouse pot experiment included 10 accessions of T. boeoticum (2n = 2x = 14, AA), originated from the different regions of the northwest and west of Iran (Table 1). A greenhouse pot experiment was performed in 2014–2015 growing season at Bu-Ali Sina University, Hamedan, Iran.
Table 1. The studied 10 T. boeoticum accessions under two different conditions
The seeds were surface sterilized by washing with 70% ethanol, followed by immersion in 5% sodium hypochlorite for 30 min and were washed three times with distilled water. In order to vernalize the seeds, they were kept in the dark condition for 21 d at 4°C before transplantation into pots. Then, five germinated seedlings from each accession with similar growth were sown into plastic pots filled with 15 kg soil comprised of agronomy-field soil (silty-loam), sand and manure in a ratio of 2:1:1. All accessions were grown at an optimal growing temperature (25 ± 3°C) and photoperiod (16/8 h light/dark cycle). The pots were arranged in a randomized complete block design with three replications in two separate experiments under non-stress (95% field capacity; FC) and drought stress (45% FC) conditions which were determined by weighing the pots. The drought stress treatment started when the seedling had approximately 4–6 leaves and continued until physiological ripening of plants. At heading time, flag leaf samples of treated and untreated plants were collected from the three replicates, rapidly frozen in liquid nitrogen, and then stored at −80°C for RNA extraction.
Measurement of the morpho-physiological traits
In the greenhouse experiment, 31 different traits related to morpho-physiology, phenology, root-characters and grain yield were measured at heading and harvest times (Table 2). In this experiment, the total water use (WU) was measured as the sum of WU during each irrigation time along plant growth stages; besides, water use efficiency (WUE) was assessed by the ratio between the economic yield per plant (EYPP) and the total WU. Leaf physiological traits, such as relative water content, excised leaf water retention (ELWR), were measured using the methods of Mguis et al. (Reference Mguis, Albouchi, Abassi, Khadhri, Ykoubi-Tej, Mahjoub, Brahim and Ouerghi2013). In addition, chlorophyll content (SPAD index) was assessed using a chlorophyll meter (SPAD-502; Konica Minolta Sensing, Inc. Osaka, Japan). At harvest time, after cutting the plants off the soil surface and separating the different parts of the plant, EYPP and other traits related to root and grain yield were evaluated on five plants in each pot.
Table 2. The information and abbreviations of 31 different measured traits in the greenhouse experiment
Data analysis for different measured traits in the greenhouse experiment
In the greenhouse experiment, in order to identify the tolerant and susceptible accessions to drought stress, statistical analysis was performed on all data from different replicates. The mean comparisons were computed using SAS v. 9.1 packages. The principal component analysis (PCA) was performed on the combined means of two conditions using Minitab v. 16 software.
Total RNA extraction and cDNA synthesis
A 100 mg of flag leaves of the most tolerant and the most susceptible accessions at the reproductive stage was used for extracting total RNA using RNX-Plus solution (CinnaGen, Tehran, Iran) according to the manufacturers' instructions. NanoDrop-1000 spectrophotometer was used to check the purification and concentration of each sample. One per cent agarose gel electrophoresis was applied to prove the integrity of isolated RNA. High-quality RNA (OD 260/230 > 2) was applied for further steps. Following the manufacturer's guideline of the Prime-Script RT reagent Kit (Takara, Japan), the first-strand cDNA was made using 2 µg of RNA.
Analysis of TaNAC2 and TaNAC69-1 TFs expression
Relative gene expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, Reference Livak and Schmittgen2001), using the following formula:
$$ \eqalign{\Delta \Delta {\rm Ct} = ({\rm Ct},\;{\rm target}\ndash {\rm Ct},\;GAPDH)_{{\rm stress}\;{\rm condition}} \cr \quad\ndash ({\rm Ct},\;{\rm target}\ndash {\rm Ct},\;GAPDH)_{{\rm non}-{\rm stress}\;{\rm condition}}.$$In a previous study, we proved GAPDH was the most stable reference gene under drought stress in wheat and was selected as the internal control gene (Jamshidi Goharrizi et al., Reference Jamshidi Goharrizi, Wilde, Amirmahani, Moemeni, Zaboli, Nazari, Moosavi and Jamalvandi2018). This reference gene produces a piece with a length of 187 bp. Forward and reverse primers for TaNAC2 (Gen Bank: AY625683.1) and TaNAC69-1 (Gen Bank: AY625682.1) were designed using the Primer3 web service (http://primer3.ut.ee/) and Gene Runner software version 6.0.28. 185 and 205 bp were considered as the production pieces' length of TaNAC2 and TaNAC69-1 TFs, respectively. A temperature of 60°C was calculated as the amplicon T m of all primers.
Forward TaNAC2, 5′-ATCAAGAAGGCCCTCGTGTT-3′
Reverse TaNAC2, 5′-TGCATCTTCTCCCACTCGTT-3′
Forward TaNAC69-1, 5′-AGTGGTACTTCTTCAGCCCC-3′
Reverse TaNAC69-1, 5′-TGCATGATCCAGTCGGTCTT-3′
Forward GAPDH, 5′-GGTGCCAAGAAGGTCATCAT-3′
Reverse GAPDH, 5′-TGGTCATCAAACCCTCAACA-3′
Rotor-Gene 3000 Real time Fast Thermo cycler (Sydney, Australia) was used to amplify the cDNA using 95°C for 35 s, then 38 cycles of 94°C for 6 and 35 s of annealing at the amplicon T m for each primer followed by a melting curve analysis. No-template controls were applied to verify that no genomic DNA existed. The attendance of an individual peak in qRT-PCR melting curve products and a single band of the expected measure in the 1.5% agarose gel after electrophoresis verified the specificity of amplicons.
Results
Identifying tolerant and susceptible accessions to drought stress
In order to assess the responses of 10 T. boeoticum accessions under non-stress and drought stress conditions, mean comparisons of them for different traits were used on the two conditions (Table 3).
Table 3. Mean comparison of different traits in 10 T. boeoticum accessions under non-stress and drought stress conditions
For each row, values with the same letter indicate no-significant differences at 5% level.
Based on the results of the mean comparison for different accessions (Table 3), the differences between accessions were significant (P < 0.05) for all phenological, morpho-physiological and root-related traits. These results indicate that there is a suitable level of genetic diversity in the studied accessions. In the studied germplasm, B5 accession significantly (P < 0.05) showed the highest mean values of EYPP and most morpho-physiological traits, including plant height (PH), peduncle length (PEL), main spike length (MSL), fertile spike number per plant (FSNPP), spikelet number per spike (SNPS), seed number per main spike (SNPMS), seed number per plant (SNPP), main spike weight (MSW), seed weight per main spike (SWPMS), biological yield per plant (BYPP), leaf area index (LAI) and WUE (Table 3). On the other hand, B5 accession significantly (P < 0.05) showed the lowest mean values of the phenological traits, including days to heading (DTH), days to anthesis (DTA) and days to maturity (DTM), together with WU and root to shoot dry weight ratio (RDW/SDW) (Table 3). Unlike the favourable B5 accession, unfavourable B6 accession had the highest amount for WU, phenological traits and the allocation of photosynthetic materials to its underground parts (root-related traits) (Table 3).
In order to evaluate accessions based on all phenological, morpho-physiological and root-related traits, and to identify the most tolerant and susceptible accessions to drought stress, PCA was performed on combined data from non-stress and drought stress treatment. Based on the results of PCA analysis (online Supplementary Table S1), the first three principal components accounted for 74.3% of the total variation of the data. By the bi-plot resulting from the PCA (Fig. 1), the studied traits and accessions were distinguished in different groups. The results (Table 3 and Fig. 1) revealed that the traits located in the first bi-plot area, such as SNPS, FSNPP, SNPMS, SNPP, BYPP, MSL, SWPMS, MSW, LAI, WUE and PH, were positively associated with EYPP, so B5 accession with high levels of these traits was known as a tolerant accession to drought stress. On the other hand, the area opposite to the first area of the bi-plot (third area) was identified as an undesirable area. B6 and B1 accessions placed around this area of the bi-plot. They were known susceptible accessions to drought stress. Since B6 accession had the lowest amount of EYPP, WUE and grain filling period (GFP) and the highest amounts of ELWR and WU. B6 accession was known as the most susceptible accession to drought stress. In addition, the mean value of this accession for root-related traits, such as root volume (RV), root fresh weight (RFW), root diameter (RD) and root area density (RAD), was higher than other accessions. The flag leaves of the tolerant and susceptible accessions to drought stress were used in molecular experiments.
Fig. 1. Bi-plot of the first (PC1) and second (PC2) principal components for 31 different traits (Table 2) of T. boeoticum accessions (B1–B10) in both non-stress and stress conditions. Accessions in the oval are tolerant (B5) and susceptible (B1 and B6) to drought stress, respectively.
TFs expression
The results of this study showed that in the most susceptible (B6) and tolerant (B5) accessions of T. boeoticum wild wheats, the TaNAC2 and TaNAC69-1 genes expression increased in response to drought stress. By applying the stress, increase in the expression of TaNAC2 and TaNAC69-1 genes in the most susceptible accession (B6) of wild wheat was very slight, but significant statistically, while in the most tolerant accession (B5) of T. boeoticum, the expressions of these two genes were very intensive, and significant statistically. TaNAC2 was up-regulated 1.66- and 9.64-fold in the most susceptible and the most tolerant accessions of T. boeoticum, respectively, by imposing drought stress. For TaNAC69-1 and under drought stress, enhancement of 1.53- and 20.12-fold was calculated for the most susceptible and the most tolerant accessions of T. boeoticum, respectively (Figs. 2 and 3).
Fig. 2. Expression of TaNAC2 gene under non-stress and drought stress conditions has been shown in (a) for susceptible accession (B6) and in the most tolerant accession (B5) in (b). Two stars indicate significance at a 99% level.
Fig. 3. Expression of TaNAC69-1 gene under non-stress and drought stress conditions has been shown in (a) for susceptible accession (B6) and in the most tolerant accession (B5) in (b). Two stars indicate significance at a 99% level.
Discussion
Morpho-physiological responses of T. boeoticum accessions to drought stress
The significant differences (P < 0.05) between the studied accessions indicate these accessions have a high level of genetic diversity, so they can provide useful information in the studies of tolerance to drought stress and breeding programmes. In line with our results, high levels of genetic diversity in Iranian accessions of T. boeoticum collected from west of Iran have been reported (Naghavi et al., Reference Naghavi, Malaki, Alizadeh, Pirseiedi and Mardi2010). Since one of the major limitations to improve drought stress tolerance in bread wheat cultivars is the narrow genetic variation, the high genetic diversity of wild relatives of bread wheat can be effective (Ashraf, Reference Ashraf2010) and help the future programmes to produce suitable cultivars. In a study on different T. boeoticum accessions collected from the northwest to the southwest of Iran, the significant differences were reported among different accessions for most agro-morphological characters (Pour-Aboughadareh et al., Reference Pour-Aboughadareh, Mahmoudi, Moghaddam, Ahmadi, Mehrabi and Alavikia2017). Indeed, a high degree of variation of morpho-physiological, phenological and yield characters is mainly related to geo-graphical origin (Mguis et al., Reference Mguis, Brahim, Albouchi, Yakkoubi-Tej, Mahjoub and Ouerghi2008). The bi-plot resulting from PCA (Fig. 1), as a statistical tool, was used to characterize and evaluate accessions based on multiple traits and to determine tolerant and susceptible accessions. In previous researches, PCA was used for grouping wild relatives of wheat based on various morphological, physiological and root-related traits (Mguis et al., Reference Mguis, Brahim, Albouchi, Yakkoubi-Tej, Mahjoub and Ouerghi2008; Pour-Aboughadareh et al., Reference Pour-Aboughadareh, Mahmoudi, Moghaddam, Ahmadi, Mehrabi and Alavikia2017; Nazari et al., Reference Nazari, Moosavi and Maleki2018). In the present study, the results of PCA analysis (Fig. 1) and the mean values of all morpho-physiological and root-related traits (Table 3) showed B5 accession (originated from Zaghe, Lorestan, Iran) was the most tolerant accession to drought stress and less affected by drought stress. B5 accession had a relatively short growth period, as the mean value of phenological traits of DTH, DTA and DTM was low in this accession. It seems this accession used the escape mechanism to improve its grain yield in response to drought stress. According to the results for B5 accession (Table 3, Fig. 1), the highest mean value of EYPP and BYPP and the lowest mean value of the RDW/SDW indicate any attempt for increasing the above-ground traits can lead to direct or indirect grain yield improvement in this wild wheat. Based on PC1 and PC2 that showed largest negative and positive loading values (online Supplementary Table S1) with the studied traits and mainly distinguished the traits in different groups, the first area of the bi-plot included the best traits and accessions (Fig. 1). In the bi-plot graph, vectors of traits with angles less than 90° are positively associated, whereas with angles higher than 90°, they are negatively associated and the intensity of the correlation is increased for angles near 0° and 180°. Also, the amount of variation described by each trait can be determined by the vector length (Lopes et al., Reference Lopes, Reynolds, Jalal-Kamali, Moussa, Feltaous, Tahir, Barma, Vargas, Mannes and Baum2012). Indeed, the traits located in the first bi-plot area, which are positively associated with economic yield, are suitable indices for selection of suitable accessions and for indirect selection to improve grain yield. In this study, WUE revealed a perfect positive relationship with economic yield (Fig. 1). In addition, considering WUE is often an important determinant of yield under stress and even as a component of crop drought resistance (Blum, Reference Blum2009); therefore, this physiological trait was suggested as a remarkable character in increasing the yield of tolerant accession. By contrast, the results of evaluation of T. boeoticum accessions by various traits (Table 3, Fig. 1) showed B6 accession (originated from Marivan, Kordestan, Iran) was the most susceptible accession in the studied germplasm. It seems B6 accession with the higher mean values of root-related or under-ground traits attributed its biomass to the roots instead of the shoots. Since the third area of the bi-plot, opposite to the first area, was determined as undesirable area, this area included the traits that are negatively associated with economic yield and with other traits that are positively associated with economic yield (Fig. 1). Therefore, B6 accession that was placed in the undesirable bi-plot area was known as the most susceptible accession to drought stress. Meanwhile, the highest WU and the lowest WUE belonged to this susceptible accession.
Expression of TFs under non-stress and drought stress conditions
In this study by applying the drought stress, changes in the TaNAC2 and TaNAC69-1 TFs expression were observed in the most susceptible (B6) and the most tolerant (B5) accessions of T. boeoticum wild wheat. A little change, but significant statistically, in the expression of these two TFs was calculated under drought stress in the most susceptible accession of T. boeoticum, while the expression of these genes was very intensive and significant statistically in the most tolerant accession of this wild wheat. A study in 2006 has shown that under drought stress, the expression of TaNAC69-1, TaNAC69-2 and TaNAC69-3 TFs was markedly up-regulated in both leaves and roots (Xue et al., Reference Xue, Bower, McIntyre, Riding, Kazan and Shorter2006). Another article in 2012 showed that TaNAC2 is also involved in response to salt, cold, drought and abscisic acid treatment (Mao et al., Reference Mao, Zhang, Qian, Li, Zhao and Jing2012). The same article demonstrated that in Arabidopsis, TaNAC2 overexpression results in enhanced tolerance to salt, freezing and drought stresses (Mao et al., Reference Mao, Zhang, Qian, Li, Zhao and Jing2012). Also, TaNAC2 overexpression results in increased expression of abiotic tension-response genes and many physiological indexes (Mao et al., Reference Mao, Zhang, Qian, Li, Zhao and Jing2012).
NAC TFs have a plant-specific NAC DNA-binding domain (Aida et al., Reference Aida, Ishida, Fukaki, Fujisawa and Tasaka1997). A previous study in 2010 proved that a wide number of NAC proteins exists in plant genomes (117 NAC genes in Arabidopsis and 151 genes in rice) (Nuruzzaman et al., Reference Nuruzzaman, Manimekalai, Sharoni, Satoh, Kondoh, Ooka and Kikuchi2010). Several researches showed that many abiotic tension-up-regulated NAC proteins adjust tension-inducible genes (Fujita et al., Reference Fujita, Fujita, Maruyama, Seki, Hiratsu, Ohme-Takagi, Tran, Yamaguchi-Shinozaki and Shinozaki2004; Hu et al., Reference Hu, Dai, Yao, Xiao, Li, Zhang and Xiong2006, Reference Hu, You, Fang, Zhu, Qi and Xiong2008). Abiotic tension-up-regulated NAC proteins are clustered into the SNAC category (Nuruzzaman et al., Reference Nuruzzaman, Manimekalai, Sharoni, Satoh, Kondoh, Ooka and Kikuchi2010), and commonly bind to a core (C/T) ACG motif (Fujita et al., Reference Fujita, Fujita, Maruyama, Seki, Hiratsu, Ohme-Takagi, Tran, Yamaguchi-Shinozaki and Shinozaki2004). The majority of abiotic tension-up-regulated NAC proteins act as transcriptional activators as illustrated in protoplast or yeast systems (Fujita et al., Reference Fujita, Fujita, Maruyama, Seki, Hiratsu, Ohme-Takagi, Tran, Yamaguchi-Shinozaki and Shinozaki2004; Lu et al., Reference Lu, Ying, Zhang, Shi, Song, Wang and Li2012). A few abiotic tension-up-regulated NAC proteins have been illustrated to act as transcriptional repressors in cells of plant (Sakuraba et al., Reference Sakuraba, Kim, Han, Lee and Paek2015).
Most likely and since the expression of TaNAC2 and TaNAC69-1 genes affect tension-inducible genes and resistance-related genes, the high amount of TaNAC2 and TaNAC69-1 TFs expression in this study in the most tolerant accession of T. boeoticum wild wheat could assist in drought tolerance of this genotype.
Conclusion
In this study, phenological, morpho-physiological and root-related responses of 10 T. boeoticum accessions, originated from eco-geographical regions of the northwest and west of Iran, were evaluated under non-stress and drought stress conditions. Statistical analysis for studied traits showed significant differences among accessions. The existence of a high level of genetic diversity in these studied accessions revealed the great potential of this germplasm may provide significant information for breeding purposes. Overall, based on the results of the mean comparison of different accessions and PCA, B5 accession (originated from Zaghe, Lorestan, Iran) and B6 accession (originated from Marivan, Kordestan, Iran) were determined as the most tolerant and susceptible to drought stress, respectively. Based on the results of expression changes in TFs, it can be concluded that TaNAC2 and TaNAC69-1 TFs are involved in response to drought stress, and could assist in drought tolerance in T. boeoticum wild wheat. These results may contribute to the existing knowledge on classical and modern breeding programmes in future. In addition, expression of TaNAC2 and TaNAC69-1 TFs can be considered as drought resistance index in wild wheats and maybe in other plants.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S1479262119000303.
Acknowledgement
We are thankful to the Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran due to providing a part of the laboratory facilities of this research. The present study was supported by Bu-Ali Sina University (Hamedan, Iran) with Grant No. 941-94 and Grant Code 4096.
Conflict of interest
The authors declare that they have no conflict of interests.
