Development and Characterization of Synthetic Hexaploid Wheat for Improving the Resistance of Common Wheat to Leaf Rust and Heat Stress

Abstract

Synthetic hexaploid wheat (SHW) is a valuable resource for breeding because it possesses more desirable traits, such as better yield and abiotic and biotic stress tolerance than common wheat. In this study, our group developed a SHW line, named ‘SynDT’, which has markedly better characteristics than Korean bread wheat ‘Keumkang’. The SynDT line is thermotolerant as it rapidly expresses heat shock proteins under heat stress. In addition, this line exhibits resistance to leaf rust by inducing the expression of antifungal enzymes, mainly chitinase, along with the rapid and high expression of pathogen-related genes. Moreover, it possesses the favorable traits of its parent wheat lines Triticum durum #24 and Aegilops tauschii #52. Therefore, the SynDT wheat line can be used as a breeding material for improving local common wheat cultivars.

1. Introduction

Hexaploid common wheat or bread wheat (Triticum aestivum L.; hexaploid genome = AABBDD) is a crucial cereal crop worldwide. A third of the global population depends on this crop, and the rise in global population has led to an increased demand for bread wheat. However, sustainable wheat production is threatened as wheat productivity has not improved significantly over time [1]. This challenge can be explained by the narrow genetic diversity of bread wheat, which is the result of a bottleneck event during its evolution, as only a few accessions of the donor species were involved in its origin and evolution [2]. Synthetic hexaploid wheat (SHW) has been artificially developed to address this issue. To mimic the natural hybridization of bread wheat, tetraploid Triticum turgidum L. wheat (2n = 4x = 28, AABB) and diploid Aegilops tauschii Coss. (2n = 2x = 14, DD) were selectively crossed to pass the elite genes of desirable traits to their progenies [2]. Consequently, SHW is a valuable source of genetic variability, with favorable traits, such as improved yield and abiotic and biotic stress tolerance, which are the primary targets of modern breeding strategies [3]. A previous study on genetic diversity in SHW has reported that this breed has higher levels of diverse amplified fragment length polymorphisms (39%) than common bread wheat (12–21%) [4]. To date, China is one of the successful countries to have managed to completely utilize SHW as a genetic resource. The well-known representative SHW line, “Chuanmai 42”, has a high average yield, and its synthetic derivative lines (SDLs) are commonly used in wheat breeding [5].

Leaf rust (LR) is a common disease in wheat caused by the fungi Puccinia triticina [6]. The fungi usually attack the leaf blades and cause a decrease in kernel weights and numbers per head. LR disease in the early stage of growth results in 30% yield loss [7]. SHW is a genetically variable source of LR resistance. “Syn137” (68.111/RGB-U//Ward/3/A. tauschii [WX629]) is resistant to LR at the seedling stage owing to the presence of a dominant LR resistance gene on chromosome 5D [8]. Moreover, “Synthetic 45” is resistant to 20 pathotypes of LR at the seedling stage. Genetic mapping has shown that the resistant gene, LrSyn45, is located on chromosome 1D [9]. A novel LR-resistant gene, Lr32, has also been identified in synthetic wheat [5]. Therefore, developing LR-resistant SHW is one of the main strategies for crop improvement.

Climate change has caused an increase in global temperature, adversely affecting plant growth. In plants, heat stress (HS) is known to cause irreversible damage when the temperature exceeds threshold levels [10]. HS negatively affects all stages of plant development [11]. Additionally, wheat is sensitive to HS. It is estimated that an increase in temperature of 1 °C causes a 6% reduction in wheat production [12]. Thus, developing SHW resistant to HS is essential. Advanced wheat lines derived from SHW have shown 13% higher yield under HS conditions than their parent lines [13]. Multiple synthetic derivatives have also shown high tolerance to HS [14].

Despite the outstanding tolerance of SHW to environmental stresses, it still processes undesirable agronomic traits, making it unsuitable for commercial usage [9]. Thus, these desirable traits of SHW are usually transferred to domesticated bread wheat to generate SDLs for the improvement of local common wheat cultivars. Many SDLs that have been registered worldwide show higher genetic diversity than their parent lines [9]. In this study, for common wheat cultivar improvement, SHW lines that can confer resistance to HS and/or LR were developed. These lines can be used as breeding materials for improving local cultivars. Our findings showed that, in comparison to Keumkang, a bread wheat cultivar that is widely grown in Korea, the SHW line developed in the present study (named SynDT) was tolerant to HS and LR. In the future, this SHW line can be crossed with local bread wheat cultivar for improving stress tolerance.

2. Materials and Methods

2.1. Plant Materials

The SHW lines, SynDT #1 and #2, are the progeny of artificial crossing between T. durum #24 (T. turgidum LEUCURUM 33) and Ae. tauschii #52 (Ae. tauschii TA 1616). Both T. durum #24 and Ae. tauschii #52 were obtained from the International Maize and Wheat Improvement Center (CIMMYT) (Table S3). The embryo was rescued, and the plantlets were grown for several weeks in half-strength MS media before it was transferred to soil. Before the heading stage, the chromosomes were doubled by colchicine treatment (solution concentration: 0.5 mg/mL); next, the plants were grown until the grains were set. The collected grains were checked and amplified. Keumkang was used for comparison with SHW.

2.2. Confirmation of D Genome in SynDT

For crossing, T. durum were chosen as receivers and Ae. tauschii as donors. Therefore, to verify the success of pollination by confirming the presence of the D genome on the synthetic wheat line, which is inherited from Ae. tauschii, a SCAR-marker, SCS253, was utilized. This marker is linked to Lr19 gene and on 7D chromosomes. The PCR condition, which is described by Huseynova et al. (2013) [15], is used to get the SCS253 bands in SynDT line and its parent wheats.

2.3. LR Infection

The P. triticina strain, HSZ0742 (NCBI Gene Bank DQ41710.1), was provided by the Korea Research Institute of Chemical Technology.

The wheat grains were sown in soil for eight days (23 °C, 16 h light/8 h dark) before infection with P. triticina. Three pots per wheat line were planted for each replicate. The plants were infected as described in a previous study [16]. The phenotype images were acquired at 8 days postinoculation (dpi).

2.4. HS Treatment

The grains were sown in soil for six days (23 °C, 16 h light/8 h dark) before being exposed to HS. Three pots per wheat line (20 grains in each pot) were planted for each replicate. At day 6, the wheat was exposed to 45 °C temperature for 8 h and then returned to normal conditions (23 °C, 16 h light/8 h dark). At day 7 or 8, HS exposure, as performed on day 6, was repeated. Images were acquired after 4 days of recovery.

2.5. Microscopy and Rust Biomass Assay

At 8 dpi, LR samples from 5 leaves were collected for either microscopy or rust biomass assays. This was performed according to the detailed protocol described by Truong et al. (2019) [16].

2.6. Protein Extraction and Enzyme Assays: Chitinase, β-1,3-Glucanase, Peroxidase Activity Assays

Fifty milligrams of LR samples from 3 leaves were collected at 0, 6, and 24 h post-inoculation (hpi). Total protein was extracted using PRO-PREP^TM protein extraction solution (iNtRON Biotechnology). The extracted solution was utilized for three enzyme assays: chitinase, β-1,3-glucanase, and peroxidase assays. These assays were performed based on a previously reported protocol [16].

2.7. Real Time RT-PCR Analysis

For HS, 6-day-old seedlings were exposed to a temperature of 45 °C for 0, 4, and 8 h. In contrast, 8-day-old seedlings were infected with P. triticina spores at 0, 6, and 24 h. The samples from 3 leaves were collected and ground in liquid nitrogen. Total RNA was extracted using TRIzol (Ambion, Life Technologies, Carlsbad, CA, USA) solution. After incubation, the samples were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was transferred to new Eppendorf following by adding 200 µL chloroform, mixing and incubating for 3 min at room temperature. The samples were then centrifuged, and the supernatant was added isopropanol, inverting, and standing on ice for 10 min. After centrifugation, the supernatant was discarded, washed with 70% ethanol and the pellets were dried for an hour. After that, DEPC distilled water was added and incubated in 55 °C for 10 min. The quality of RNA is checked by agarose gel running. From that, cDNA is synthesized by using the Thermo Scientific RevertAid first strand cDNA synthesis kit [17]. The difference in transcript levels was determined by qRT-PCR analysis (CFX connect Real-Time PCR, Bio Rad, Hercules, California 94404, USA). The internal control for normalization was Plant actin eu97. The primers used in this study are listed in Table S1. The qRT-PCR data were analyzed by Bio-Rad CFX Manager 3.1 software.

2.8. Data Analysis

For robust results, each experiment was repeated at least twice. One set of representative data was chosen for analysis. The standard deviation (SD) value was calculated from three replicates of each treatment. Statistical Analysis System (SAS) was used to perform one-way analysis of variance (ANOVA) and Tukey’s test (α = 0.05). Among lines within a treatment condition, different letters (a, b, or c) indicate significant different based on one-way ANOVA (p < 0.05).

3. Results and Discussion

3.1. Process of Generating Synthetic Wheats and Their Morphology

In this study, more than 50 lines each of T. durum and Ae. tauschii cultivars were obtained from the CIMMYT. Preliminary screening was done to select the cultivars with at least mild tolerance to HS or LR (data not shown). Both T. durum and Ae. tauschii were planted in the field for grain amplification and crossing. For crossing, we chose T. durum as receivers and Ae. tauschii as donors. Before the grain-filling stages, we performed emasculation. The anthers inside each spikelet from T. durum were removed and covered with polythene bags. Three days after emasculation, the pollens of Ae. tauschii were transferred to the emasculated T. durum. After three weeks, we collected the synthetic grains without endosperms. The embryos of synthetic grains were rescued and placed on MS media containing NAA hormone until they were large and strong enough to grow in soil (Figure 1A). At this stage, the synthetic plants were haploid; when the plants attained maturity, we treated them with colchicine to double their chromosome number and let them grow until the kernels were set. Figure 1B illustrates the process of colchicine treatment: removing the plants from the soil, leaving small leaves, washing off the soil in roots, immersing the plant roots in colchicine solution and aeration for 6 h, rinsing with water, and replanting the treated plants back to the soil. After colchicine treatment, the spikes, and kernels of the synthetic plants were successfully set (Figure 1C). To verify our synthetic wheats, we checked the presence of the D genome from Ae. tauschii, using the SCS253 marker. Through the preliminary PCR experiments, we found that this marker is only shown bands in the Ae. Tauschii but not T. durum lines, which is a specific marker to check the presence of D genome in hexaploid wheat. As shown in Figure 1D, T. durum #24, which contains the A and B genomes, did not have any band of SCS253 marker. Only A. tauschii #52 and SynDT #1 and #2 containing the D genome showed bands at 737 bp. The confirmed synthetic wheat plants were planted in the field for grain amplification.

In this study, the responses of our synthetic wheat (SynDT) to LR and HS were compared to those of Keumkang. In addition, we also examined T. durum #24 and Ae. tauschii #52, which are the parents of SynDT. Before studying their response to LR and HS, we also observed that the studied wheat varieties had different grain morphology and weight. SynDT had large grains, with long and thin shape. Compared to Keumkang, T. durum #24 is longer in length but similar in width. Keumkang has short and round shapes, whereas those of Ae. tauschii #52 were the smallest (Figure 2A,B). Furthermore, there is a correlation between grain size and weight. The order of weights of 100 grains/line is SynDT > Keumkang > T. durum #24 > Ae. tauschii #52 (Figure 2C, Table S4). The size of wheat grains is one of the most crucial agronomic traits because of its effect on grain weight, milling quality, and market price [18]. In addition, larger grains may have favorable traits of increased yield and seedling vigor [19]. Taken together, our synthetic wheat lines have larger and heavier grains, which is a promising trait for breeding value.

3.2. Tolerance of SynDT to HS Due to the Induction of HSPs

First, we examined the response of our synthetic wheat to HS. The 6-day-old wheat seedlings were subjected to extreme HS at 45 °C for 1, 2, or 3 consecutive days for 8 h per day. After 4 days of recovery, we observed that SynDT lines grew better than Keumkang, with full second leaves emerging from the stem. Most of the Keumkang plants were severely affected by HS, with their second leaves being damaged and growing abnormally (Figure 3A,B). Therefore, based on the growth statuses of the second leaves, we measured the recovery rates of the studied wheat lines, and found that SynDT lines have higher recovery rates than Keumkang (Figure 3C, Table S5). Keumkang was included in this study for good comparison [16] because, in a previous study, Keumkang had the highest tolerance to HS among the common Korean wheat cultivars. Additionally, we checked the response of the parent wheat, T. durum #24 and Ae. tauschii #52, to HS and found that Ae. tauschii #52 is more tolerant to HS than Keumkang and T. durum #24 (Figure S1). Therefore, we believe that the HS tolerance of SynDT lines is mainly inherited from Ae. tauschii #52. In addition, some studies have revealed that the D genome from Ae. tauschii is a valuable resource for the variability in improved agronomic traits [20,21].

At the molecular level, the response to HS is mainly characterized by the expression of HSPs. The increased accumulation of HSPs might contribute to thermotolerance [22]. Under stress conditions, HSPs play a role in protein stabilization and refolding [23]. Therefore, we examined the transcript levels of HSP17.3 (NCBI X58279.1), HSP70.1 (NCBI KJ027551.1), and HSP101b (NCBI AF097363) (Tables S2 and S6). HSP17.3 is a small HSP (sHSP) that binds to non-native proteins to prevent thermal aggregation [23]. The transcript levels of HSP17.3 were quickly induced in response to HS (Figure 4A). At the 4 h time point, HS significantly increased the expression levels of HSP17.3 in SynDT #1 compared to that in Keumkang (106-fold); HSP17.3 expression in T. durum #24 was also upregulated. At 8 h, the transcript levels of HSP17.3 were reduced in most wheat lines except in Keumkang. In a previous study, the expression of HSP17 was also induced in response to HS in “HD2329” and “C-306” wheat cultivars [24]. HSP70 belongs to one of the main families of HSPs and functions as a chaperone in protein refolding and prevents aggregation [22]. HSP70 is also involved in the response to various environmental stress factors, such as heat, cold, oxidative stress, heavy metals, and pathogens [25]. In our study, HS exposure also induced the expression of HSP70.1, particularly in SynDT #1 at 4 h (Figure 4B). At 8 h of HS, the expression levels of HSP70.1 showed no difference between Keumkang and SynDT #1. HSP70.1 expression in the parent T. durum #24 was maintained at 4 and 8 h of HS exposure. Similarly, SynDT #1 showed significantly higher HSP101b expression than Keumkang at 4 h (262-fold). There was no difference in the transcript levels of HSP101b after 8 h of HS. Moreover, the expression of HSP101b in T. durum #24 was higher at 8 h than at 4 h. In contrast, Ae. tauschii #52 had higher transcript levels of HSP101b at 4 h compared to that at 8 h. HSP101 is known to reactivate aggregated proteins [26]. In maize, the accumulation of HSP101 protein was increased in response to HS [27]. Thus, SynDT showed higher expression levels of HSPs than Keumkang, especially at 4 h, indicating that the former responded to HS more quickly than Keumkang to protect the plants from damage. Moreover, in the parent lines, HSP17.3 and HSP70.1 were mainly expressed in T. durum #24, while HSP101b was expressed in both.

3.3. Resistance of SynDT Wheat Cultivar to LR by the Induction of Chitinase and PR Genes

All 8-day-old wheat lines were infected with P. triticina. After 10 dpi, urediniospores were observed on the leaf surface of Keumkang, whereas no spores were detected on those of SynDT #1 and #2 (Figure 5A). Microscopy revealed that a mass of hyphae had developed from the infected sites in Keumkang, but not in the synthetic wheats (Figure 5B). In addition, the results of rust biomass were consistent with those observed using microscopy (Figure 5C, Table S7). We also checked the response of T. durum #24 and Ae. tauschii #52 to LR and found that T. durum #24 was mildly sensitive to LR, whereas Ae. tauschii #52 was resistant (Figure S2). Therefore, we believe that our synthetic wheats may have inherited the LR-resistant phenotype from Ae. tauschii #52.

Plants have multiple mechanisms to defend themselves from pathogenic infections. Among these, the accumulation of antifungal proteins is vital in inhibiting fungal growth. Chitinase, β-1,3-glucanase, and peroxidase are common antifungal proteins that induce LR. The first component of fungi in contact with plants is its cell wall, of which chitin is one of the fundamental components. Chitinase can hydrolyze the substrate, chitin, into N-acetylglucosamine oligomers [28,29]. We determined the chitinase levels of 8-day-old infected seedlings. Both Keumkang and SynDT cultivars showed a gradual reduction in chitinase levels at the time of LR exposure. However, at 6 and 24 hpi, the chitinase activity of the SynDT cultivar was significantly higher than that of Keumkang (1.6-fold and 1.4-fold, respectively) (Figure 6A, Table S8). Furthermore, the transcript levels of CHI8, a chitinase-encoding gene, were upregulated when Keumkang and SynDT lines were infected with LR, and SynDT showed the highest expression at 6 hpi (Figure 6B, Table S8). There was no difference in the transcript levels of CHI8 at 24 hpi, indicating that CHI8 is an early responsive gene during infection, especially in the SynDT cultivar. We also checked the transcript levels of the PR genes involved in systemic acquired resistance (SAR) [30]. Among the PR genes, PR4 encodes the chitin-binding protein [31]. We found that the expression levels of PR4 were upregulated in response to LR (Figure 6C, Table S8). At 6 hpi, the SynDT line had significantly higher PR4 levels than Keumkang. The transcript levels of PR4 in SynDT substantially increased at 24 hpi. Between parent wheats, T. durum #24 showed a greater induction of PR4 transcript levels compared to that of Ae. tauschii #52 at 6 and 24 hpi.

ß-1,3-Glucan, a substrate of β-1,3-glucanase, is another important component of the fungal cell wall. In combination with chitinase, β-1,3-glucanase catalyzes the hydrolytic cleavage of β-1,3/1,6-glucans in the cell wall [32,33]. In our study, in response to LR, the glucanase levels of Keumkang and SynDT were similar at 6 hpi but decreased in the SynDT line at 24 hpi (Figure 7A, Table S9). Moreover, the PR2 gene, which encodes proteins with β-endoglucanase activity, was more induced in SynDT at 6 and 24 hpi, compared to Keumkang. PR2 was also highly expressed in Ae. tauschii #52 (Figure 7B, Table S9). Numerous studies have shown the upregulation of PR genes in plants in response to pathogenic infections [34,35,36]. Both PR2 and PR4 are considered important proteins that can be targeted in the fungal cell wall, thereby leading to fungal death. The coaction of PR genes can increase the resistance to pathogenic attacks in plants [37]. Therefore, the upregulation of PR2 and PR4 in the SynDT line can partially explain its resistant phenotype. β-1,3-glucanase and chitinase belong to the PR2 and PR4 families, respectively. Moreover, it is known that chitinase, along with β-1,3-glucanase, can inhibit the hyphal growth of many pathogenic species [38]. Our results reconfirmed that chitinase is the primary enzyme responsible for LR resistance in the SynDT wheat cultivar.

We further checked peroxidase activity and observed that the levels of peroxidase increased gradually in response to LR in both Keumkang and SynDT, but it was slightly higher in the latter (Figure 8, Table S10). The enzyme peroxidase is crucial for the synthesis of lignin, leading to cell wall lignification, which serves as a physical barrier against infections [39]. In plants, protective levels vary, which are determined by specific enzyme activity, enzyme concentration and localization inside cells, host–pathogen interactions, and fungal characteristics [39]. Therefore, the study on defensive responses is complicated. In our study, the combination of different antifungal proteins, especially chitinase activity, could be involved in the cascade to prevent LR in SynDT cultivar.

Lastly, Lr34, which encodes the ATP-binding cassette (ABC) transporter, is a well-known LR resistance gene in many wheat cultivars [40]. Lr34 is located on chromosome 7DS and is one of the crucial resistance genes in wheat breeding because of its ability to inhibit multiple pathogenic species [39]. Our results showed that Lr34 had significantly higher expression levels in SynDT than in Keumkang (14.1-fold), particularly at 6 hpi (Figure 9, Table S11). Therefore, the fast response of Lr34 in SynDT may contribute to LR resistance.

4. Conclusions

Thus, in comparison to Keumkang, SynDT showed higher resistance to both HS and LR. SynDT is tolerant to HS, owing to the elite genes from its parents and fast-induced HSPs that protect cells from detrimental effects. SynDT also showed enhanced chitinase activity and high transcript levels of PR genes that help to cope with LR. Recently, the use of SHW in wheat breeding as a genetic resource has gained promising results for the improvement of common wheat worldwide. Furthermore, the possession of valuable genes in SynDT is critical in common wheat breeding for stress tolerance. In the future, SynDT can be used as a breeding material for local Korean bread wheat cultivars.