Data collection for different wheat cultivars

The collected data included total RNA of leaf tissues of studied plants. The libraries of the species under study contained 3 susceptible and 3 resistant genotypes. C306 is a cultivar resistant to drought, salinity and heat. C306 cultivar is derived from the cross between the cultivars RGN / CSK3 // 2 * C5 91/3 / C217 / N14 // C281 [21]. WL711 is a semi-short, high yielding, drought-sensitive, medium flowering plant with moderate germination ability. The parents of this figure are [(S308 × Chris) × Kalyansona] [9]. Y12-3 and A24-39 are resistant and sensitive to drought, respectively. Y12-3 is Yehudiyya (35 ° 42′ N; 32 ° 56′ E) which has high water productivity, yield stability and high-water use under drought stress but genotype A24-39 from Amirim (35° 27′ N); 32° 55′ E), which has high efficiency, performance stability and low water use efficiency [28]. The interpretation file for these libraries was obtained from the affymetrix site (, which contains 44 GSM in total.

Microarray meta-analysis by Rank prod package

In resistant cultivars of the wheat plant, 4% (2394/61290) of DEGs were showed decreased expression and 4.5% (2670/61290) of DEGs, presented increase expression, also revealed in susceptible cultivars that expression of 7% (4183/61290) DEGs were decreased and expression of 6% (2591/61290) DEGs were increased.

Identify similar and different genes in the DEG by Venn diagram

Venn diagrams were designed on common probes id. Wheat Venn diagram showed that in resistant cultivars were 460 probe ID down expressed and 658 probe ID up expressed. The number of decreased expression probe IDs was 2264 and the number of increased expression probe IDs was 1561 insensitive cultivars (Fig. 2).

Fig. 2
figure 2

Venn diagram based on wheat significant expression probe ids in result of meta-analysis. 2670 probe ids in tolerant and 3591 probe ids in sensitive cultivar have increase expression. 2394 probe ids in tolerant and 4182 probe ids in sensitive cultivar decrease expression

Thirteen probe IDs, including phosphoenolpyruvate carboxylase and putative calreticulin, were found to have increased expression in tolerant wheat cultivars, whereas the sensitive cultivars showed a significant decrease (Table 3). Calreticulin (CRT) is an important multifunctional protein that has been identified in many eukaryotic cells except yeast and erythrocytes [33]. At first CRT was known to be an important Ca2+ binding protein, but recent research has shown that CRT plays a role in many cellular functions such as calcium-binding, glycoprotein proper folding, interaction with cellular receptors, RNA binding activity and interaction with the immune response [37]. In higher plants phosphoenolpyruvate carboxylase (PEPC) is a cytosolic enzyme and there are several isoforms. PEPC is widely distributed in green algae and bacteria, too [45]. Stoma opening, fruit ripening, and seed maturation are different functions for these enzymes. Some of C3 strains have been genetically modified to produce more PEPC [34].

Table 3 Probe ids that have dissimilar DEGs expression in sensitive and tolerant cultivars

Thirty probe IDs such as carboxypeptidase D and chlorophyll a-b binding protein showed significantly high expression in sensitive cultivars but decreased in resistant cultivars (Table 3). In a similar pathway that is the carboxypepteptase-D-arginine-nitric oxide (CPD-Arg-NO) pathway, estrogen, androgen, and PRL (prolactin) cause cell survival. CPD membrane-bound metalloproteinase and membrane activity secrete arginine and lysine C-terminal lysine. Further, the CPD acts on the Golgi transport network to process polypeptides/pro-hormones that generate the secretory pathway. CPD exists in plasma membranes [49]. The CPD-plasma membrane acts on extracellular substrates and the arginine are released by CPD transfers to cells that are the common substrates of the two enzymes, arginase, and nitric oxide synthase. Photosynthesis is important for the plant’s development, which involves the collection of light and the transfer of solar energy using the chlorophyll a/b light absorption (LHC) proteins. LHC proteins are made from abundant thylakoids encoded by nuclear genes. In higher plants, the LHC protein contains a large 10–12 member gene family and the peripheral light receptors consist of photosystem I (PSI) and photosystem II (PSII) [12], the helices are linked by an ion pair. Each helix binds to chlorophyll molecules (chlorophyll a and b) and some carotenoids in the thylakoid membrane. This type of binding is needed for solar energy transmission, light absorption, and light protection [27].

Identification different expression transcription factor (TF)

Transcription factors play an important role in regulating all processes of a plant’s life [32]. Some types of TFBS elements elaborate in response to different environmental stresses contain MYB, WRKY, HSF, and C2H2 [20]. In this study, 40 transcription factors that identified based on the genes present in the meta-analysis results, showed significant expression changes (Fig. 3). In tolerant and sensitive cultivars, seven transcription factors including MADS-box TaAGL35, drought-responsive factor-like, DRFL1a zinc-finger showed increased expression. Four transcription factors including bZIP, MADS-box TaAGL11, MIKC-type MADS-box, bZip type bZIP1, and WM19A had decreased expression in both tolerant and sensitive cultivars. In resistant cultivars, Myb3, ethylene-responsive 5a, MIKC-type MADS-box WM24B, and salinity inducible ERF4 transcription factors increased expression and two transcription factors, EREBP and NAC NAM, showed a significant decrease in expression. Five transcription factors MYB, WRKY15, MADS-box TaAGL8, WRKY39, increased expression in sensitive cultivars and only one transcription factor, WRKY45, showed a significant decrease in expression. These TFs play different roles in regulating metabolism and plant behavior in different conditions, which is an important reason why they have shown different behavior between susceptible and resistant cultivars.

Fig. 3
figure 3

Significant expression Transcription factor genes in result of meta-analysis

Gene ontology characterization in each susceptible and resistant plants

Gene ontology classification of differential expressed genes was performed separately, in susceptible and drought-resistant cultivars. This classification is a common way to interpret transcriptom data as a first step in functional genomic analysis. Study of ontology of expressed genes in three main categories including biological processes, cellular components, and molecular function were classified using REVIGO site as follows.

Gene ontology refers to the modified genes of susceptible plants

In the molecular function group, expressed genes divided into six categories: glucosyltransferase activity, UDP-glucosyltransferase activity, nutrient reservoir activity, transferase activity, transferring hexosyl groups, transferase activity, transferring glycosyl groups, and transcription factor activity, sequence-specific DNA binding. The highest number of genes was in the category of transcription factor activity and the lowest number of genes was in the category of glucosyltransferase activity.

In biological processes group, expressed altered genes divided into 22 categories. the highest number of genes was in the cellular process category with 150 number in input list and the lowest in the SCF-dependent proteasomal ubiquitin-dependent protein catabolic process, proteasomal protein catabolic process and proteasomal ubiquitin-dependent protein catabolic process with 5. The cellular group includes the smallest category. The expressed genes in this subgroup are divided into two categories. The ubiquitin ligase complex and SCF are ubiquitin ligase complex.

Gene ontology related to the modified genes of resistant plants

The gene ontology of altered genes expressed in resistant cultivars was divided into two groups of molecular function and biological process. In the biological subgroup, the altered genes were in the response to stress category and in the molecular subgroup, the genes were in the nutrient reservoir activity category.

Gene network of drought resistance

Network structure and subnet analysis were done, according to the Protein-protein interaction (PPI) dataset downloaded from STRING. The resulting PPI network was 180 proteins have been investigated, 106 proteins are related to proteins of expressed genes in sensitive wheat cultivars and 74 proteins are related to proteins of resistant cultivar genes. The giant component which included the majority of the entire network protein containing 74 nodes in resistant cultivars and 106 nodes in sensitive cultivars (Fig. 4).

Fig. 4
figure 4

Gene network for the wheat, sensitive cultivars (a) and tolerant cultivars (b). If the line is not connected to the protein, it does not indicate the association between that protein and other proteins. The number of lines further indicates that there is a large association between that protein and other existing proteins

Hub genes of drought resistance: PEPC and TaSAG7

Cytoscape software has been used to identify effective genes among meta-analysis datasets. It is a popular platform for analyzing biological networks [41]. Twenty-five hub genes, using the MCC method based on the correlation of gene expression limited to probes that have gene names from the meta-analysis results, have been identified for sensitive and tolerant cultivars individually. The results showed that in sensitive cultivars of ribosomal protein P1, glutathione transporter, SPP2 and also for tolerant varieties of vascular pyrophosphate H+, myo protein VIIIA1, glucose transporter protein and wpk4 protein kinase were highly ranked (Fig. 5) (Tables 4 and 5). The RPP1 ribosomal protein is one of three phosphoproteins in the large 60S subunit of the eukaryotic ribosome. RPLP1 plays an important role in the long-term phase of protein synthesis. RPLP1 provides a heterodimer with RPLP2 dimers [7]. The glutathione transferase gene (GSTs; also known as glutathione S-transferase) are the major stage II detoxification enzymes mainly found in the cytosol. In addition to the role of enzymes in catalyzing the binding of electrophilic substrates to glutathione (GSH), they also perform other functions. They have peroxidase and isomerase activities, which can inhibit N-terminal June kinase (thus cells protected from H2O2-induced cell death) [40]. Sucrose is an essential carbohydrate for plants and other photosynthetic organisms and is known as one of the major photosynthetic products. The SPP2 gene catalyzes sucrose biosynthesis in the final step. SPP encoding genes have been described in various plant species including Arabidopsis, tomato, rice, wheat, corn, and coffee. Four genes show homology to SPP in Arabidopsis, whereas three and four genes are described in wheat and rice, respectively [1]. Blastn analysis by NCBI site was performed for the two selection genes in rice and Arabidopsis plants. The results show PEPC sequence in Arabidopsis has 95.12% identity sequence and Accession is X98080. TaSAG7 sequence in Arabidopsis has 72.62% identity sequence and Accession is AK316978. PEPC sequence in rice has 84.48% identity sequence and Accession is CP056060. TaSAG7 sequence in rice has 88.76% identity sequence and Accession is XM_015789456.

Fig. 5
figure 5

Cytoscope diagram based on the symbols gene. A Resistant cultivars. B Sensitive cultivars

The ranking is based on the MCC method in cytoHubba. Red to yellow, respectively, indicates a higher to lower rank

Table 4 Hub genes identified for susceptible varieties
Table 5 Hub genes identified for resistant varieties

KEGG pathways and heat maps

Isocitrate lyase (ICL) plays an important role in the metabolic processes of citric, methylcitric, and glyoxylate cycles [39], which is the bypassed pathway of the TCA cycle that converts isocitrate to glyoxylate and succinate. During germination, ICL plays a key role in lipid-sugar conversion using the acetyl unit from acetyl-CoA in arabidopsis, the product of β-oxidation, by the glyoxylate cycle and gluconeogenesis [10] ICL is a single-copy gene in both rice [31] and Arabidopsis [50]. ICL and malate synthase were involved in the transfer of leaf peroxisomes to glycosystems and this process was correlated with aging and senescence [55]. Phosphvanol pyruvate carboxylase (PEPC) is a cytosolic enzyme in higher plants and is also widely distributed in green algae and bacteria. In higher plants, there are several PEPC isoforms. These enzymes are involved in a variety of functions, including stomatal opening, fruit ripening, and grain maturity. To date, several C3 species have been genetically engineered to produce more PEPC [42]. The KEGG pathway identified for isocitrate lyase and PEPC genes have played an important role in resistance to drought stress in wheat (Fig. 6). The expression heat map of the identified genes is shown expression difference in susceptible and resistant gene profiles [54]. (Fig. 7, Table 3).

Fig. 6
figure 6

KEGG pathway maps of glyoxylate and dicarboxylate metabolism and pyruvate metabolism. TaSAG7 and PEPC genes are active in these two pathways, which are important genes in drought tolerance

Fig. 7
figure 7

Heat map of DEGs in the resistant and susceptible wheat cultivars. The red bars indicate increases and the blue bars indicate decrease expression genes

Laboratory studies of PEPC and TaSAG7 genes by qPCR

We used qPCR to investigate the expression change of two selected genes at four-point time, control, 2, 4, and 7 days after cessation of irrigation in leaf tissue of different wheat cultivars (Figs. 8 and 9). The amount of RWC for 2 days of stress decreased for two tolerant cultivars 6O4, 3% and Sirvan 13% and also for Sundor cultivar 2% and Tajan 15%. The amount of RWC for 4 days of stress decreased for two tolerant cultivars 6O4, 15% and Sirvan 31% and also for susceptible cultivars Sundor 25% and Tajan 24% and finally the amount of RWC for 7 days of stress decreased for two tolerant cultivars 6O4, 48% and Sirvan 48% and also for susceptible cultivars Sundor 46% and Tajan 52%. Numerous studies have reported the negative effects of increased PEPC activity on Pn or biomass. For example, production of C4 PEPC in rice led to a decrease in photosynthesis due to increased respiration under light conditions and severely reduced growth [43]. Other studies have shown that transgenic plants expressing PEPC have relatively high biomass under stress conditions such as optical oxidation, heat, and drought. C4 PEPC has been cloned and identified in many crops including rice, wheat and Arabidopsis. Expression of maize PEPC gene in transgenic rice plant has increased antioxidant capacity under drought stress [42]. PEPC maize gene expression in transgenic rice plant increases antioxidant capacity under stress conditions such as light oxidation, heat, and drought [15]. In general, based on the meta-analysis, PEPC gene increased expression by 1.37 in resistant cultivars and decreased by − 1.30 in susceptible cultivars. The expression of this gene was expected to increase in Sirvan and 6O4 cultivars and in two cultivars. Tajan and Sundor dropped. By examining the mean comparisons made in this study, the results obtained from qPCR were consistent with the results of meta-analysis (Figs. 10 and 11).

Fig. 8
figure 8

Expression level of PEPC gene in tolerant wheat (6O4 and Sirvan) and sensitive wheat (Sundor and Tajan) genotypes at three stress levels 2, 4, and 7 days after drought stress. FC column based on the results of meta-analysis performed for susceptible and resistant cultivars placed separately next to each expression level columns for better comparison of the results

Fig. 9
figure 9

Expression level of TaSAG7 gene in tolerant wheat (6O4 and Sirvan) and Sensitive wheat (Sundor and Tajan) genotypes at three stress levels 2, 4, and 7 days after drought stress. Based on the results of meta-analysis, this gene has increased only in resistant cultivars. For convenience of comparison, the FC column obtained from the meta-analysis placed next to the resistant cultivars

Fig. 10
figure 10

Real-time-PCR (RT-PCR) of PEPC gene under normal conditions and drought stress. Bands 1–4 show RT-PCR under the normal conditions and bands 5–8 show RT-PCR under drought stress

Fig. 11
figure 11

Real-time-PCR (RT-PCR) of PEPC gene under normal conditions and drought stress. Bands1–2 and 5–6 show RT-PCR for the normal conditions, while bands 3–4 and 7–8 show RT-PCR under drought stress

During biotic and abiotic stresses, the activity of plant defense systems against free radicals decreases and the production of oxygen free radicals increases. This destroys cell membranes through peroxidation of phospholipids and stops intracellular activities, especially enzymatic reactions. Providing a carbon skeleton for carbohydrate synthesis is not the only role of the glycoxylate cycle. This pathway has also been shown to play an anaplastic role in microorganisms and plants. The glyoxylate cycle can play this vital role through the net production of succinate from acetyl coa [2]. The two enzymes lyase isolate and malate synthetase are unique to the glyoxylate cycle, which avoids the decarboxylation steps of the Krebs cycle. Based on a meta-analysis of TaSAG7 gene in resistant cultivars, a 2.14-fold increase in expression has been reported, which results from real-time PCR are consistent with the results of meta-analysis and confirm the result of meta-analysis.

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