Analysis of wheat gene expression related to the oxidative stress response and signal transduction under short-term osmotic stress

Water shortage is a major environmental stress that causes the generation of reactive oxygen species (ROS). The increase in ROS production induces molecular responses, which are key factors in determining the level of plant tolerance to stresses, including drought. The aim of this study was to determine the expression levels of genes encoding MAPKs (MAPK3 and MAPK6), antioxidant enzymes (CAT, APX and GPX) and enzymes involved in proline biosynthesis (P5CS and P5CR) in Triticum aestivum L. seedlings in response to short-term drought conditions. A series of wheat intervarietal substitution lines (ISCSLs) obtained by the substitution of single chromosomes from a drought-sensitive cultivar into the genetic background of a drought-tolerant cultivar was used. This source material allowed the chromosomal localization of the genetic elements involved in the response to the analyzed stress factor (drought). The results indicated that the initial plant response to drought stress resulted notably in changes in the expression of MAPK6 and CAT and both the P5CS and P5CR genes. Our results showed that the substitution of chromosomes 3B, 5A, 7B and 7D had the greatest impact on the expression level of all tested genes, which indicates that they contain genetic elements that have a significant function in controlling tolerance to water deficits in the wheat genome.


Expression of genes encoding enzymes involved in ROS signaling (MAPK3 and MAPK6). The
MAPK3 and MAPK6 genes showed two different patterns of expression in the tested T. aestivum L. seedlings exposed to drought (Fig. 1a,b). TaMAPK3 transcript levels significantly decreased after 6 hours of exposure in most of the tested lines. However, different expression profiles were observed after 1 and 3 hours of stress for individual lines. Lines containing chromosomes of homoeologous groups 3 (3A, 3B, 3D), 4B, 6B and 7B showed a rapid response and immediate reduction in MAPK3 expression after 1 hour of PEG treatment. The downregulation of MAPK3 was noticed at subsequent time points (after 3 and 6 hours). Only 3D and 4B showed a transient increase in MAPK3 expression after 3 hours. A significant induction of MAPK3 was detected in the first hours of stress for the drought-tolerant cultivar S29 and lines 1D, 4A, 5D, 6A, followed by a rapid decrease in transcript levels. For the remaining forms, no MAPK3 induction was observed in the first hours of plant exposure to drought, and then a significant decrease occurred. The biggest differences in the MAPK3 expression pattern compared to the recipient were detected for lines 3A, 3B, 6B and 7B. The drought-sensitive cultivar JP showed a decrease in the transcript level after 3 hours. A similar expression pattern was recorded for 1A, 5A and 7D. MAPK3 expression in lines 4D, 5B and 5D decreased only after 6 hours of water shortage. The results of the study indicated the weakest changes in MAPK3 expression for lines containing chromosomes of homoeologous group 2 (2A, 2B, 2D) under the conditions tested (Fig. 2a).
Most ISCSLs showed an increase in the transcript level of MAPK6 after 6 hours. However, different expression patterns were observed during stress treatment for individual lines. Rapid MAPK6 induction already occurred after 1 hour in the drought-tolerant cultivar S29. Similar expression profiles were recorded for lines involving chromosomes of homoeologous group 2 (2A, 2B, 2D), 4D, 6B, 3D, 4A and 5D. A significant increase in MAPK6 transcript levels was detected for those lines after 3 hours of 10% PEG exposure. Lines 3A, 3B, 5A, 6A and 7B showed the most significant changes in MAPK6 expression compared to the recipient. The drought-sensitive cultivar JP exhibited a delayed response, and MAPK6 expression was elevated significantly only after 3 and 6 hours. A similar delayed induction of MAPK6 was noted for lines 4B, 5B and 7D. Most lines with the substitution of A genome chromosomes (1A, 3A, 5A) showed no significant changes in MAPK6 expression throughout the 6 hours of stress (Fig. 2b).
www.nature.com/scientificreports www.nature.com/scientificreports/ expression of genes encoding antioxidant enzymes (CAt, ApX and GpX). The overall pattern of CAT expression was a significant increase in transcript levels after 1 hour, which remained stable after 3 and 6 hours of stress (Fig. 3a). This expression trend was observed for lines with the substitution of A genome chromosomes (1A, 2A, 3A, 4A) and 3D, 5B and 6B forms. However, S29, ISCSLs with substitution of chromosomes from groups 2 (2B, 2D), 4 (4A, 4D), 7 (7B, 7D) and lines 1D, 5D and 6A initially showed an increase in CAT expression, followed by a decrease after 6 hours of drought. This expression pattern was characteristic for lines with the substitution of D genome chromosomes. The most significant alterations in CAT gene expression compared to the recipient (S29) were observed for lines 3B, 4B and 5A, that response was similar to the drought-sensitive cultivar JP (Fig. 4a).
The analysis of the GPX gene indicated different expression patterns in the tested plants (Fig. 3c). S29 showed a significant increase in expression after 3 hours, followed by a decrease during prolonged drought (6 hours). A similar trend was observed for lines 1D, 2B, 4B, 5D and 6A. The substitution of chromosomes 3A, 3D, 5A, 5B, 6B, 7B and 7D had the greatest impact on GPX expression compared to the recipient. Continuous upregulation of GPX expression was recorded in lines with the substitution of A genome chromosomes (1A, 2A, 4A). Some of the tested lines exhibited a significant decrease in GPX expression during 10% PEG treatment: lines containing chromosomes of homoeologous groups 5 (5A, 5B) and 7 (7B, 7D) and line 6B. The transient downregulation of the GPX gene occurred in the first hours, followed by an increase in the transcript level for the majority of lines with the substitution of D genome chromosomes (2D, 3D, 4D, 7D). No significant changes of GPX expression were observed for the drought-sensitive cultivar JP (Fig. 4c).
Expression of genes involved in proline biosynthesis (P5CS and P5CR). The results of the study indicated a significant increase in the expression of both genes: P5CS and P5CR. However, elevated transcript levels of the P5CS gene were observed immediately after 1 hour of stress, while a delayed induction (after 3 hours) was noticed for P5CR ( Fig. 5a,b).
The exposure of plants to drought resulted in a rapid response and increase in P5CS expression in most of the tested lines. S29, JP, 1D, 2B, 4B, 4D, 5D and lines with the substitution of A genome chromosomes (1A, 2A, 3A, 6A) already showed P5CS upregulation after 1 hour of stress. Moreover, the level of transcript for the drought-tolerant cultivar was significantly higher than that for the drought-sensitive one at each time point. A www.nature.com/scientificreports www.nature.com/scientificreports/ delayed response was observed for lines 3D, 4A and 6B (after 3 hours) and for 2D, 5A, 5B, 7B and 7D (after 6 hours of stress). Therefore, the results of the analysis showed that lines containing chromosomes from homoeologous groups 1, 2 and 4 exhibited a fast response to drought and continuous upregulation of P5CS, while a delayed reaction to stress was observed in lines containing chromosomes from homoeologous groups 5 and 7. Line 3B did not show any changes in P5CS gene expression. The transient downregulation of the P5CS gene was found in lines 2D and 7B after 1 hour (Fig. 6a).
The results showed similar trends in P5CR and P5CS regulation for the majority of the tested lines. A significant increase in P5CR expression was detected in S29 and JP after 1 hour of stress however, the upregulation of P5CR was higher in the drought-tolerant cultivar S29 than that in drought-sensitive JP. Similar observations were made for lines 1D, 2A, 2B, 4B, 5D and 6A. A delayed response to water stress, manifested as P5CR gene upregulation, was found in lines 4A, 4D, 7D (after 3 hours) and 2D, 5B, 6B (after 6 hours). A significant decrease in the P5CR transcript level was observed for 2D, 3D, 4A and 7B after 1 hour of drought. In comparison to the recipient (S29), lines 3B, 3D, 5A, 7B and 7D showed the highest changes in the P5CS and P5CR expression profile in response to drought (Fig. 6b).

Discussion
Plant MAPK cascades have been intensively studied as a mechanism involved in the regulation of stress response. Many studies have indicated the important roles of MAPK3 and MAPK6 in plant cells 33,41,52,53 . However, the information available on MAPKs in T. aestivum is still limited 33,[35][36][37] .
This study tested genes encoding two MAPKs (MAPK3 and MAPK6), three antioxidant enzymes (CAT, APX and GPX) and two enzymes involved in proline biosynthesis (P5CS and P5CR). According to our data, the main pattern for the MAPK6 gene was increased expression in drought-treated plants. MAPK6 gene induction during water deficiency has also been reported in wheat 35 and A. thaliana 32 . However, some of the tested lines did not show any changes in MAPK6 expression (1A, 3A, 5A, 6A, 3B and 7B), which had also been demonstrated in wheat cultivar Chinese Spring using RNA-seq 36 . The downregulation of MAPK3 gene expression was observed in all www.nature.com/scientificreports www.nature.com/scientificreports/ tested lines. The results suggest that kinase encoded by this gene may also function in signal transduction but as negative regulator A decrease in MAPK3 expression was described for wheat under phosphorus deprivation 35 and for Cucumis sativus L. under drought conditions 54 . However, our analysis of the MAPK3 gene indicated different expression patterns for each line during 6 hours of PEG treatment. Based on previous data, we suggest that there is no unequivocal evidence for a specific MAPK3 gene expression pattern under drought. Wen et al. 35 . reported no significant changes in MAPK3 expression in wheat, while Zhan et al. 36 . observed an increase in the TaMAPK3 transcript level. MAPK3 expression upregulation was also described in A. thaliana 55,56 . Wang et al. 40 . reported a high level of ZmMAPK3 expression after 1 hour of 10% PEG treatment and decreased expression after 6 hours. These authors suggest that there are common mechanisms underlying the abiotic stress signaling and convergent points that include the production of H 2 O 2 generated by stress stimuli and the reliance on common signaling cascades. The versatile system also allows linking the H 2 O 2 signal to the MAPK cascade and target genes. Identifying all cascade modules and broadening our knowledge about the regulation of all molecular mechanisms under stress conditions will provide further insight into the biological response of plants. Different profiles of MAPK3 and MAPK6 expression obtained in this study suggest that these kinases are regulated differently. Obtained results showed that MAPK6 overexpression is one of the components of plant response to drought, whereas MAPK3 do not play a role as a positive signal transduction regulator during this kind of stress. Moreover, a reverse reaction of MAPK3 versus MAPK6 expression in experimental conditions can indicate on the regulation mechanism based on feedback between these two kinases. Analysis based on ISCSLs revealed that chromosomes 3A, 3B, 6B, and 7B as well as 3A, 3B, 5A, 6A, and 7B contained genes involved in MAPK3 and MAPK6 expression regulation, respectively (Table 1). These results could provide a premise that chromosomes 3A, 3B and 7B contain genetic elements responsible for controlling of both analyzed kinases. www.nature.com/scientificreports www.nature.com/scientificreports/ Water deficiency leads to ROS formation and the induction of genes that encode antioxidant enzymes. The involvement of those genes in promoting plant responses to unfavorable conditions has been well established in many studies [57][58][59] . It has been reported that the overexpression of genes encoding antioxidant enzymes causes higher tolerance to stress factors in A. thaliana 60 and rice 16,61 . However, some data indicate that retaining stable gene expression can also confer drought tolerance in plants 62,63 .
In this study, the significant increase in CAT expression was observed in the majority of tested lines. CAT gene upregulation was also observed in wheat subjected to mild drought for 7 hours 64 , in Macrotyloma uniflorum , and GPX (c) genes in S29(JP) substitution lines after 1, 3 and 6 h of 10% PEG treatment. Bars represent standard deviation. The expression level for non-exposed plants was used as calibrator (relative expression level = 1), *change significant at the 0.05 level. All samples were analyzed in three full biological and three technical replications.
www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ after 78 hours 65 and in barley after 2 days of drought 66 . High levels of CAT expression were observed in Cleome gynandra and Cleome spinosa during prolonged drought exposure (10 days) 67 . However, in this study, some of the tested lines showed a significant decrease in CAT expression after 6 hours of water stress. Previous studies have revealed variable responses to different durations and severities of drought stress in various plant species. A lower CAT transcript level was observed in wheat after 10 days 68 or in barley after 9 and 16 days of stress 66 . Our results suggest that short-term drought increased CAT expression as a rapid plant response. However, prolonged exposure to drought could lead to the inhibition of expression and the reduction in transcript levels. PEG (10%) did not alter CAT expression in JP and lines 3B, 4B, and 5A, what can indicate the presence of encoding or regulatory elements on these chromosomes (Table 1). Similar observations were reported for Poa pratensis 57,62 and Koeleria macrantha 53 . Moreover, in this work, we report similar expression patterns for MAPK6 and CAT genes. Xing et al. 69 . suggested that MAPK6, as a component of a cascade involved in signal transduction, mediates H 2 O 2 formation and CAT expression changes. Our results can support this hypothesis and indicate, that both enzymes are involved in the complex immediate reaction to osmotic stress in wheat.
Our analysis of the genes encoding two peroxidases showed that no constitutive expression pattern can be defined for all analyzed lines (especially for GPX). One of the potential factors responsible for this situation is fact that both of these enzymes are encoded by numerous sequences localized in different parts of the wheat genome. The trend of response to examined stress revealed enhancement of both genes expression, however, the time shift between them was observed. For APX the quick response was noticed, whereas for GPX a slower building up of the response was shown.
APX gene showed its expression downregulation in lines 3B, 5A, 7B and 7D. A reduction in APX transcript level was reported previously for Arachis hypogaea L 70 . and in roots of P. pratensis under drought treatment conditions 62 . No significant changes were found in the leaves of P. pratensis, which is consistent with our results obtained for lines 2D, 3D, 4B and 5B. The upregulation of the APX gene was observed in lines with the substitution of A genome chromosomes (2A, 3A, 4A, 6A), lines 2B, 5D, 6B and the drought-tolerant cultivar S29. Many previous studies have pointed to an increase in APX expression in different plant species under drought conditions, including Pisum sativum L. 71 , K. macrantha 63 , P. pratensis 57 and Hordeum vulgare L. 66 . We observed a higher APX expression level in S29 compared than that in JP. This outcome has also been reported in drought-tolerant and -sensitive barley under water shortage conditions 66 . Some data indicate that APX gene overexpression enhances tolerance to drought in transgenic rice 53 and tobacco 58 .
Our results of GPX expression analysis are consistent with those of previous studies performed on K. macrantha 63 or P. pratensis 57,62 . GPX upregulation was detected in some of the tested lines. The induction of the GPX gene can be the result of higher H 2 O 2 levels in plant cells or reduced enzyme activities 72 . An increase in GPX expression was also recorded for K. macrantha 63 , a drought-tolerant cultivar of T. aestivum 68 , and in wheat roots 72 . Our study showed a significantly elevated level of GPX expression in S29. This phenomenon has been previously reported for other tolerant T. aestivym cultivars after 10 days of drought 68 .
The results of our research based on ISCSLs suggest that genetic elements encoding and/or regulating the genetic activity of ascorbate peroxidase and guaiacol peroxidase may be present notably on chromosomes belonging to homoeologous groups 3, 5, and 7 ( Table 1).
The current results for antioxidative system have demonstrated that catalase and ascorbate peroxidase, which can efficiently scavenge H 2 O 2 and prevent its accumulation to toxic levels, are major antioxidant enzymes. However, these enzymes have different affinities for H 2 O 2 and play different roles in scavenging. CAT does not need a reductant to scavenge H 2 O 2 , making it reducing power-free, whereas APX needs a reductant (ascorbate) 62,66,73 . According to our results, the CAT expression pattern was similar to APX in most of the tested lines (S29, JP, 1D, 2A, 2B, 3A, 4A, 4B, 5A, 5D, 6A, 6B). Transcript levels significantly increased in these forms after 1, 3 and/or 6 hours of stress. The results of the analysis indicated that this expression pattern was characteristic for lines with the substitution of A genome chromosomes and lines containing chromosomes of homoeologous groups 2, 4, 5 and 6. Similar regulation of CAT and APX expression could be explained by the fact that proteins encoded by these genes are both involved in the scavenging of H 2 O 2 produced during oxidative stress, and their gene expression is likely regulated in the same way. Bian and Jiang 62 suggested that CAT and APX may facilitate efficient H 2 O 2 scavenging in leaf cells, although CAT and APX have different affinities for H 2 O 2 . No relationship was found between the expression of these two genes in the remaining lines. Different expression patterns have been described for tobacco plants exposed to drought, where an increase in Cat3 and a decrease in strAPX expression occur 74 . MAPK3  3A, 3B, 6B, 7B   MAPK6  3A, 3B, 5A, 6A, 7B   CAT  3B, 4B, 5A   APX  1A, 2D, 3B, 3D, 4B, 5A, 5B, 7B, 7D   GPX  3A, 5A, 5B, 6B, 7B, 7D   P5CS  3B, 3D, 5A, 7B, 7D   P5CR 3B, 3D, 5A, 7B, 7D www.nature.com/scientificreports www.nature.com/scientificreports/ Proline is synthesized in a two-step process catalyzed by ∆ 1 -pyrroline-5-carboxylate synthetase (P5CS), followed by the reduction of P5C to proline by ∆ 1 -pyrroline-5-carboxylate reductase (P5CR) 19 . Both genes showed a significant increase in expression levels in the tested wheat substitution lines with 10% PEG treatment. P5CS and P5CR upregulation was also found in K. macrantha under water stress conditions 63 . Previous results show that increased expression of these genes may control plant responses and contribute to a higher level of RWC and proline accumulation or lead to a decrease in MDA content. The overexpression of genes involved in proline synthesis enhances drought tolerance in transgenic Glycine max 75 , Petunia hybrida 76 , Cicer arietinum 77 and T. aestivum 25 . Our study demonstrated that the exposure of plants to 10% PEG resulted in a rapid response reflected by an immediate increase in P5CS and P5CR expression levels in the first hours of stress. This observation confirms the hypothesis, that the major mechanism underlying proline biosynthesis in response to drought is regulation at the transcriptional level. This mechanism has also been reported in other plant species under drought conditions. An increase in P5CS transcript levels was reported for Oryza sativa L. after 2 and 5 hours 78 , Brassica napus after 1, 2 and 6 hours 22 and for A. thaliana after 5 and 12 hours of water stress 79 . P5CS gene upregulation has also been observed under osmotic stress conditions induced by NaCl in wheat 19 , moth bean (Vigna aconifolia) 80 , soybean 81 and A. thaliana 82 . An increase in P5CR expression was described in P. sativum 83 and A. thaliana 84 after 6 and 12 hours of NaCl treatment, respectively. Our results showed a higher level of P5CS expression in the drought-tolerant cultivar compared to that in the drought-sensitive one, which has also been found in rice 78 . In most of the tested lines, the P5CS gene showed an immediate induction after 1 hour of stress, while changes in P5CR were observed after 3 or 6 hours. These results suggest that the P5CS gene plays a key role in proline biosynthesis and the plant response to short-term drought. Similar expression patterns were reported for A. thaliana. Changes in AtP5CS gene expression were detected after 2 hours and in AtP5CR after 24 hours of osmotic stress 82 . These results can indicate that product of P5CS gene could induce P5CR expression as well. Analysis of the results obtained for each ISCSL indicates, that genes encoding enzymes associated with proline biosynthesis (P5CS and P5CR) or their regulators were located on chromosomes 3B, 3D, 5A, 7B, and 7D ( Table 1). The location of these genes on 3B and 3D chromosome is supported by data from Phytozome (Table 2) however, the remaining genome regions indicated need further investigations. Moreover, lines with the substitution of the 3A and 4A chromosomes showed very strong upregulation of both proline biosynthesis genes expression after 6 hours of the experiment. This observation can indicate the presence of regulatory elements on these chromosomes.

Gene Chromosome
In conclusion, our study shed light on the molecular mechanisms of plant responses to short-term drought conditions. The first reaction of the tested wheat lines was characterized by changes in the transcript levels of CAT, a gene encoding an enzyme closely associated with H 2 O 2 detoxification. Furthermore, stress induced the expression of genes involved in proline biosynthesis and the MAPK6-mediated signaling pathway. The results suggest that plant responses are controlled by differential gene expression regulation.
In the present study, we identified chromosomes associated with the initial wheat response to short-term stress using a set of S29 substitution lines (JP) with varying drought tolerance. The data indicated that the substitution of chromosomes 3B, 5A, 7B and 7D had the largest impact on the expression level of all tested genes and could play a critical function in controlling tolerance to water deficits in the wheat genome (Table 1). Moreover, we suggest that structural or regulatory genes involved in the first plant response to drought may be located on those chromosomes. Our results are consistent with previous data reported by several research groups [85][86][87][88] .
Further investigations should be performed, including gene expression analysis of other MAPKs and antioxidant enzymes as well as evaluation of their activity to gain new insight into drought tolerance in plants and to clarify the roles of these enzymes in the general pattern of the stress response. A better understanding of the mechanisms underlying drought tolerance will help to develop stress-tolerant genotypes. Moreover, mapping strategies should also be devised for the localization of genetic loci associated with wheat responses to drought. Grains were sterilized with chlorine gas for 4 hours and transferred to Petri dishes containing filter paper soaked in distilled water. For germination induction, the grains were incubated at 4 °C for 48 hours and germinated for 2 days in the dark at 24 °C. Germinating seedlings were placed in plastic pots containing MS medium and maintained in a hydroponic culture in a growth chamber for 5 days under control conditions (light/dark regime of 16/8 h at 25 ± 3 °C, relative humidity of 50 ± 10%, light intensity during the daytime was 350 μmol m −2 s −1 ). For stress induction polyethylene glycol (PEG) addition to the medium was used. PEG cause osmotic stress occurrence, inducing plant water deficit similar to drought condition 89 . The main advantage of this experimental model is fact, that hydroponic culture provides a constantly reduced water potential, which is not possible in soil models 90 . Drought stress was induced after 5 days of acclimatization by applying 10% PEG-6000 dissolved in MS medium solution. To avoid hypoxia occurrence, the medium was aerated consistently. Wheat seedlings were collected after 1, 3 and 6 hours of stress treatment. Plants growing in MS medium without PEG were used as a control.

Methods
Identification of analyzed genes transcripts and the detection system design. This study analyzed genes encoding antioxidant enzymes (CAT, APX, GPX), enzymes involved in proline biosynthesis (P5CS and P5CR) and two MAPKs (MAPK3 and MAPK6). For genes encoding antioxidant enzymes and enzymes involved in proline biosynthesis the Phytozome 12.1 (https://phytozome.jgi.doe.gov) database was used to identify full-length cDNA sequences within the wheat genome (Triticum aestivum v. 2.2) ( Table 2) 91 . For identified (2019) 9:2743 | https://doi.org/10.1038/s41598-019-39154-w www.nature.com/scientificreports www.nature.com/scientificreports/ sequences, multiple sequence alignment was performed using T-Coffee software with M-Coffee algorithm 92 and the representative consensus sequence was built. For analyzed MAPKs the sufficient information about coding sequences was not present in Phytozome database. Because of that fact we decided to identify wheat ortologs of A. thaliana MAPK3 and MAPK6 genes in NCBI GenBank database. Based on the alignment results, the accessions AF079318 and AY173962 were selected for MAPK3 and MAPK6, respectively. In order to confirm the homology of these sequences with respective MAPK3 and MAPK6 sequences from other selected plant species (A. thaliana, Brachypodium distachyon, O. sativa and Zea mays) an alignment was performed using BLAST tool (https://blast.ncbi.nlm.nih.gov) ( Table 3). Obtained sequences were used as templates for detection system design. Gene-specific primers and probes were designed using PrimerBLAST software (https://www.ncbi. nlm.nih.gov/tools/primer-blast/) 93 (Tables 4 and 5). For qPCR dual-labeled (6-FAM as a reporter, BHQ-1 as a quencher) TaqMan probes were applied. extraction of total RNA and qpCR (Real-time pCR) analyses. Total RNA from wheat seedlings was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The quality and quantity of isolated RNA were evaluated by 2% agarose gel electrophoresis and spectrophotometrically using DeNovix DS-11. Total RNA (1.5 µg) was used to synthesize cDNAs using the iScript ™ cDNA synthesis kit (Bio-Rad). Quantitative PCR was performed with TaqMan probes. PCRs were carried out in a total volume of 20 µl containing 80 ng of cDNA, 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 800 nM of each primer and 250 nM of TaqMan probe. Standard curves were generated from five dilution points for each primer pair. In the first step, amplification was started at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of PCRs: 95 °C for 15 s and 60 °C for 1 min. For qPCR analyses the Quant Studio3 system (ThermoFisher Scientific) was used.
The T. aestivum cDNA clone whv16n3n16 5-(GenBank accession number: CJ705892) was used as an internal standard for normalization of qPCR results, as it revealed the most stable expression in the tested wheat lines under experimental treatment conditions. In order to determine the best reference for qPCR, ten potential reference genes were selected: 5 of them were 'typical' reference genes (actin, tubulin, GAPDH, ubiquitin, and  www.nature.com/scientificreports www.nature.com/scientificreports/ translation elongation factor) and the remaining 5 were selected from Genevestigator database using RefGenes tool. The validation of the reference genes was performed with 4 different algorithms: geNorm, NormFinder, BestKeeper, and RefFinder (data unpublished). Relative gene expression was calculated using the 2 −∆∆Ct method 94 . Each sample was analyzed in three full biological and three technical replicates. For determination of the specificity of qPCR reaction NTC (No Template Control) was applied for each reaction. The results were analyzed using the dedicated relative quantification software module from ThermoFisher Cloud (ThermoFisher Scientific). statistical analyses. In order to determine statistical significance of the change of analyzed genes expression in comparison to respective control forms a one-way ANOVA with Dunnett's post-hoc test was performed at 0.05 probability level based on the ∆C T values. All statistical analyses were performed using Statistica 13.1 software package (Dell Inc.).

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.