Genome-Wide Identiﬁcation and Expression Proﬁling of Pathogenesis-Related Protein 1 ( PR-1 ) Genes in Durum Wheat ( Triticum durum Desf.)

: Pathogen-related proteins (PRs) are diversiﬁed proteins with a low molecular weight implicated in plant response to biotic and abiotic stress as well in regulating different functions in plant maturation. Interestingly, no systematical study has been conducted in durum wheat ( Triticum turgidum subsp. durum ). In the present study, 12 PR-1 genes encoding a CAP superfamily domain were identiﬁed in the genome of Triticum turgidum subsp. durum , which is an important cereal, using in silico approaches. Additionally, phylogenetic analysis showed that the PR-1 genes were classiﬁed into three groups based on their isoelectric point and the conserved motif domain. Moreover, our analysis showed that most of the TdPR-1 proteins presented an N-terminal signal peptide. Expression patterns analysis showed that the PR-1 gene family presented temporal and spatial speciﬁcity and was induced by different abiotic stresses. This is the ﬁrst report describing the genome-scale analysis of the durum wheat PR-1 gene family, and these data will help further study the roles of PR-1 genes during stress responses, leading to crop improvement.


Introduction
Plants grow under constantly varying biotic and abiotic (salty soils, drought, waterlogging, UV lights, extreme temperatures, heavy metals, and many others) stress conditions. These factors are detrimental to plant maturation as they cause many metabolic perturbances and are the main cause of cell death [1,2]. To overcome such constraints, plants have evolved different mechanisms such as the accumulation of different phytohormones (abscisic acid (ABA), ethylene (ET), jasmonic acid (JA), methyl jasmonate (MeJA) and salicylic acid (SA), phytoalexins, and different defense genes (antioxidants, Mitogen Activatd proteins Kinase). These proteins control the accumulation of small proteins known as Pathogen-related proteins (PR proteins) which inhibit pathogen multiplication in uninfected plant organs [3].
PRs are a group of proteins presenting low molecular weight that accumulate in plants during plant maturation, gene expression, flowering, plasmolysis, and senescence [4,5]. Moreover, several studies have shown that PR proteins are accumulated following pathogen infection. They control plant production of different peptides, proteins, or compounds which prevent pathogen infections where they start or act as toxins to pathogens [6]. As a result, plants increase their tolerance to protect themselves against pathogens [5]. In addition, PR proteins respond to various types of abiotic stresses such as drought, freezing, UV-lights, salinity, light, and osmotic stress [7,8] and hormones such as salicylic acid (SA) [9], abscissic acid (ABA), jasmonic acid (JA), and auxin (IAA) [8,10,11]. The PR proteins are thermostable, protease-resistant proteins that have a molecular weight of~5-43 kDa. In addition, the PR-genes are expressed in all plant organs. Interestingly,

Identification, Distribution, Gene Structures, and Conserved Motifs of PR-1 in Triticum durum
After using BLASTp v 2.13.0 searches, the verification of the possession of CAP domain (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1) (Figure 1), and the elimination of redundant sequences, twelve TdPR1 sequences were obtained from the Triticum durum genome (named TdPR1.1 to TdPR1.12). Moreover, the analyses of the exon-intron organization were performed to understand the evolution of the TdPR1 genes. We found that the selected PR-1 genes shared the same structure. In fact, only one gene (TdPR1.12) had two exons and one intron, whereas the other 11 TdPR1 genes possessed a single exon. Furthermore, no 5 -UTR or 3 -UTR region was detected in the gene sequences ( Figure 1). By using the Multiple Em for Motif Elicitation (MEME) (version 5.5.1), five motifs were identified. Four of them were presented in all the selected TdPR1 proteins (Motif 1, 2, 3, and 4), whereas motif 5 (presented by red boxes) was missing in TdPR1.5 ( Figure 1). gene (TdPR1.12) had two exons and one intron, whereas the other 11 TdPR1 genes possessed a single exon. Furthermore, no 5′-UTR or 3′-UTR region was detected in the gene sequences ( Figure 1). By using the Multiple Em for Motif Elicitation (MEME) (version 5.5.1), five motifs were identified. Four of them were presented in all the selected TdPR1 proteins (Motif 1, 2, 3, and 4), whereas motif 5 (presented by red boxes) was missing in TdPR1.5 ( Figure 1). The genomic features of TdPR1 members are presented in Table 1. The distribution of TdPR-1 genes were on five different chromosomes. Two genes were located on Chr5A, Chr7A, and Chr7B, whereas only one gene was located on chromosome 6B. The rest (about a third of the selected genes) were clustered on chromosome 5B ( Figure 2, Table 1). The genomic features of TdPR1 members are presented in Table 1. The distribution of TdPR-1 genes were on five different chromosomes. Two genes were located on Chr5A, Chr7A, and Chr7B, whereas only one gene was located on chromosome 6B. The rest (about a third of the selected genes) were clustered on chromosome 5B ( Figure 2, Table 1).

Multiple Alignment and Phylogenetic Relationship among the TdPR1 Genes
In order to understand the evolutional history of the candidate's proteins, a phylogenetic tree was reconstructed. TdPR-1 genes were distributed into three groups: Group I comprising genes encoding all acid proteins, Group III included five basic proteins and the six-basic protein, and the only TdPR1.5 located on Chr6B were clustered in Group II. Our findings show that this gene did not have the conserved motif 5 and had a different cleavage site sequence CNA-AF (Table 2; Figure 3). Chromosomal distribution of PR-1 genes in Triticum durum genome, based on their groups I, II, and III. Gene ID are colored in orange, green, and blue, respectively, by using MG2C server v2.1.

Multiple Alignment and Phylogenetic Relationship among the TdPR1 Genes
In order to understand the evolutional history of the candidate's proteins, a phylogenetic tree was reconstructed. TdPR-1 genes were distributed into three groups: Group I comprising genes encoding all acid proteins, Group III included five basic proteins and the six-basic protein, and the only TdPR1.5 located on Chr6B were clustered in Group II. Our findings show that this gene did not have the conserved motif 5 and had a different cleavage site sequence CNA-AF (Table 2; Figure 3). Based on the multiple alignment performed by the Muscle algorithm, we found that TdPR1 protein sequences are homologous. Indeed, the analysis indicated the presence of the Pfam0018 domain (CAP) in all the TdPR 1 proteins, (shown by red lines in the Figure 4). In addition, the blue box indicates the presence of the signal peptide (SP) regions (located at the N-terminal portion of the proteins; 24-31; Table 2). These peptides were found in all identified TdPR1 proteins except for TdPR1.3 and TdPR1.4. Moreover, only TdPR1.5 showed a cleavage site (between two alanine residues), whereas the others presented a common cleavage site between alanine (A) and glutamine (Q) amino-acid residues ( Table 2). Based on the multiple alignment performed by the Muscle algorithm, we found that TdPR1 protein sequences are homologous. Indeed, the analysis indicated the presence of the Pfam0018 domain (CAP) in all the TdPR 1 proteins, (shown by red lines in the Figure  4). In addition, the blue box indicates the presence of the signal peptide (SP) regions (located at the N-terminal portion of the proteins; 24-31; Table 2). These peptides were found in all identified TdPR1 proteins except for TdPR1.3 and TdPR1.4. Moreover, only TdPR1.5 showed a cleavage site (between two alanine residues), whereas the others presented a common cleavage site between alanine (A) and glutamine (Q) amino-acid residues (Table 2). According to the results given by the PROSITE database, TdPR1 sequences contain two conserved domain CRISPs (cysteine rich secretory proteins) in the C-terminal: CRISP family signature 1 (CRISP_1) (shown by black box Figure 4;) and CRISP family signature 2 (CRISP_2) (shown as pink box Figure 4; Supplemental Figure S1).

Identification of Putative Zn 2+ and CaM Binding Domains
We have recently identified a CaM binding domain located at the C-terminal part of the protein that ensures the interaction of the CaM/Ca 2+ complex with the TdPR1 protein [31]. To investigate whether other identified TdPR1s harbor such a domain, we analyzed the structure of the 12 TdPR1 using the calmodulin target database. As revealed in Figure  5, only TdPR1.2, TdPR1.3, and TdPR1.5 harbor calmodulin binding domains at the C-terminal portion of the protein, whereas all the other TdPR1 proteins do not have such domains in their structures ( Figure 5). According to the results given by the PROSITE database, TdPR1 sequences contain two conserved domain CRISPs (cysteine rich secretory proteins) in the C-terminal: CRISP family signature 1 (CRISP_1) (shown by black box Figure 4;) and CRISP family signature 2 (CRISP_2) (shown as pink box Figure 4; Supplemental Figure S1).

Identification of Putative Zn 2+ and CaM Binding Domains
We have recently identified a CaM binding domain located at the C-terminal part of the protein that ensures the interaction of the CaM/Ca 2+ complex with the TdPR1 protein [31]. To investigate whether other identified TdPR1s harbor such a domain, we analyzed the structure of the 12 TdPR1 using the calmodulin target database. As revealed in Figure 5, only TdPR1.2, TdPR1.3, and TdPR1.5 harbor calmodulin binding domains at the C-terminal portion of the protein, whereas all the other TdPR1 proteins do not have such domains in their structures ( Figure 5).

Identification of Putative Zn 2+ and CaM Binding Domains
We have recently identified a CaM binding domain located at the C-terminal part of the protein that ensures the interaction of the CaM/Ca 2+ complex with the TdPR1 protein [31]. To investigate whether other identified TdPR1s harbor such a domain, we analyzed the structure of the 12 TdPR1 using the calmodulin target database. As revealed in Figure  5, only TdPR1.2, TdPR1.3, and TdPR1.5 harbor calmodulin binding domains at the C-terminal portion of the protein, whereas all the other TdPR1 proteins do not have such domains in their structures ( Figure 5).  On the other hand, COACH analysis revealed the presence of three putative zinc ligand binding sites in the TdPR1.2 protein. In fact, ion (Zn 2+ ) and a zinc molecule EAH ((5S, 7E, 9E, 11Z, 14Z)-5-hydroxyicosa-7, 9, 11,14-tetraenoic acid) were predicted. Different amino acids are implicated in this binding as revealed by the server which are 73 and 121 aa for the Zn ion (PDB Hit: 3mz8A) ( Figure 6A) and 35,39,42,83,95,96,99,100,103,121,125,150 amino acids implicated in a possible interaction with the molecule EAH (PDB Hit: 3u3uC; Figure 6B). On the other hand, COACH analysis revealed the presence of three putative zinc ligand binding sites in the TdPR1.2 protein. In fact, ion (Zn 2+ ) and a zinc molecule EAH ((5S, 7E, 9E, 11Z, 14Z)-5-hydroxyicosa-7, 9, 11,14-tetraenoic acid) were predicted. Different amino acids are implicated in this binding as revealed by the server which are 73 and 121 aa for the Zn ion (PDB Hit: 3mz8A) ( Figure  6A) and 35,39,42,83,95,96,99,100,103,121,125,150 amino acids implicated in a possible interaction with the molecule EAH (PDB Hit: 3u3uC; Figure 6B).

Physicochemical Properties
Several analyses showed the different characteristics of the TdPR1 proteins listed in Table 3. In fact, the amino acid lengths of different proteins were between 164 and 207 aa with a molecular weight (MW) for all proteins ranging from 17 to 19 kDa. Among these proteins, 50% are acidic (pI < 7) and the others are basic (pI > 7). The negative GRAVY index (Grand average of hydropathicity) indicates that all TdPR1 proteins are non-polar (hydrophilic) proteins [50]. In addition, the thermostability parameter of proteins is measured by the aliphatic index (AI). The high aliphatic index of TdPR1 proteins, ranging from 64.46 to 76.76, indicates that they are thermo-stable under a wide temperature range [51].

Physicochemical Properties
Several analyses showed the different characteristics of the TdPR1 proteins listed in Table 3. In fact, the amino acid lengths of different proteins were between 164 and 207 aa with a molecular weight (MW) for all proteins ranging from 17 to 19 kDa. Among these proteins, 50% are acidic (pI < 7) and the others are basic (pI > 7). The negative GRAVY index (Grand average of hydropathicity) indicates that all TdPR1 proteins are non-polar (hydrophilic) proteins [50]. In addition, the thermostability parameter of proteins is measured by the aliphatic index (AI). The high aliphatic index of TdPR1 proteins, ranging from 64.46 to 76.76, indicates that they are thermo-stable under a wide temperature range [51].

Prediction of Transmembrane Helices on TdPR1
The topological analysis shows the existence of the transmembrane helixes in all the TdPR1 proteins, except TdPR1.7. In fact, the three online tools indicated the presence of transmembrane domains in TdPR1.2; TdPR1.5; TdPR1.9, and TdPR1. 10. The results also showed that 66.66% of the online servers predicted that TdPR1.1, TdPR1.3, TdPR1.4, TdPR1.6, TdPR1.8, and TdPR1.12 have the transmembrane domain, whereas one out of three online tools indicated that this transmembrane helix is present in TdPR1.11 with a percentage of 33.33 (Table 4).

Predicted Secondary and 3D Structures of TdPR-1 Proteins
Secondary structures of proteins were predicted using the SOPMA server. The percentage of α-helices, extended strand, β turn, and random coil were 30.92-40, 14.59-18.08, Plants 2023, 12, 1998 9 of 23 2.44-6.78, and 40.36-48.17, respectively. Structural conformation of a protein can occurs via protein phosphorylation by adding the phosphate groups to serine, threonine, or tyrosine residues [52]. Using the NetPhos 3.1 server, phosphorylation sites were predicted in TdPR1 proteins and ranged between 11 and 22 ( Figure 7). The predicted channel number found in the studied TdPR1 proteins were registered between 0 and 16 ( Figure 7). TdPR1.10 VAI23305.

Predicted Secondary and 3D Structures of TdPR-1 Proteins
Secondary structures of proteins were predicted using the SOPMA server. The percentage of α-helices, extended strand, β turn, and random coil were 30.92-40, 14.59-18.08, 2.44-6.78, and 40.36-48.17, respectively. Structural conformation of a protein can occurs via protein phosphorylation by adding the phosphate groups to serine, threonine, or tyrosine residues [52]. Using the NetPhos 3.1 server, phosphorylation sites were predicted in TdPR1 proteins and ranged between 11 and 22 ( Figure 7). The predicted channel number found in the studied TdPR1 proteins were registered between 0 and 16 ( Figure 7).

Figure 7.
Percentage occurrence of the secondary structure analyses of TdPR-1 proteins. The different conformations of TdPR-1 proteins: α-helices, extended strands, β turns, and random coils were provided by SOPMA tool. BetaCavityWeb server was used to identify the putative numbers of channels. Predicted phosphorylation sites (PPS) were identified by NetPhos 3.1 server. PR-1 proteins were identified by different colors.
The predicted 3D structures of the TdPR1 proteins were constructed using the alpha fold online server. The 3D TdPR-1 proteins models presented four α-helices and several 34 . Percentage occurrence of the secondary structure analyses of TdPR-1 proteins. The different conformations of TdPR-1 proteins: α-helices, extended strands, β turns, and random coils were provided by SOPMA tool. BetaCavityWeb server was used to identify the putative numbers of channels. Predicted phosphorylation sites (PPS) were identified by NetPhos 3.1 server. PR-1 proteins were identified by different colors.
The predicted 3D structures of the TdPR1 proteins were constructed using the alpha fold online server. The 3D TdPR-1 proteins models presented four α-helices and several antiparallel β-sheets. According to the CASTp 3.0 analysis, molecular pockets were identified in all candidates. The top three predicted pockets, with the largest volume, are indicated as red, blue, and yellow, respectively ( Figure 8).

In Silico Analysis of Cis-Elements
The cis-acting element in the TdPR1 promoters could be divided into four categories, such as development-related elements, environmental stress-related elements, hormoneresponsive elements, and transcription factor binding sites, based on the biological function analyzed by Plantcare (Figures 9 and 10). Stress responsive cis-elements were found in most of the PR-1 promoters except for TdPR1.9. Cis-regulatory elements belonging to this class are associated with drought, anaerobic anoxic specific inducible element, and low-temperature response. All TdPR1 regulatory regions have more than four cis-element hormone responses. The MeJA-and abscisic acid responsive motifs are the most abundant in most of TdPR1. Thus, ABRE (abscisic acid response elements) are the elements which had the highest motif number in the TdPR1 promoter region (12 motifs in TdPR1.8). Salicylic acid, auxin, and gibberellin responsive elements were found in some TdPR1 promoters. Moreover, different cis-acting regulatory elements involved in growth and development are present. The most abundant motifs are related to light response as all the identified TdPR1 proteins presented a diversity of light response elements. G-box is the only motif present in all the identified promoters with a maximum number registered for TdPR1.8 (11 G-boxes). In addition, endosperm expression, seed-specific regulation, meristem expression, and circadian control are found in the majority of PR-1 in Triticum durum. The binding domains of a single TF family, MYB, were identified in four, eight, nine, and twelve TdPR1 promoters.
antiparallel β-sheets. According to the CASTp 3.0 analysis, molecular pockets were identified in all candidates. The top three predicted pockets, with the largest volume, are indicated as red, blue, and yellow, respectively ( Figure 8).

In Silico Analysis of Cis-Elements
The cis-acting element in the TdPR1 promoters could be divided into four categories, such as development-related elements, environmental stress-related elements, hormone-responsive elements, and transcription factor binding sites, based on the biological function analyzed by Plantcare (Figures 9 and 10). Stress responsive cis-elements were found in most of the PR-1 promoters except for TdPR1.9. Cis-regulatory elements belonging to this class are associated with drought, anaerobic anoxic specific inducible element, and low-temperature response. All TdPR1 regulatory regions have more than four cis-element hormone responses. The MeJA-and abscisic acid responsive motifs are the most abundant in most of TdPR1. Thus, ABRE (abscisic acid response elements) are the elements which had the highest motif number in the TdPR1 promoter region (12 motifs in TdPR1.8). Salicylic acid, auxin, and gibberellin responsive elements were found in some TdPR1 promoters. Moreover, different cis-acting regulatory elements involved in growth and development are present. The most abundant motifs are related to light response as all the identified TdPR1 proteins presented a diversity of light response elements. G-box is the only motif present in all the identified promoters with a maximum number registered for TdPR1.8 (11 G-boxes). In addition, endosperm expression, seed-specific regulation, meristem expression, and circadian control are found in the majority of PR-1 in Triticum durum. The binding domains of a single TF family, MYB, were identified in four, eight, nine, and twelve TdPR1 promoters.

Predicting TdPR1 Pproteins Subcellular Localization
The subcellular localization of TdPR1 proteins performed by the online tool WoLF PSORT and represented by a heatmap graphic using Tbtools v1.108 are shown in Figure  11. Indeed, with a high level of prediction, 10 TdPR1 proteins were found to be located in the extracellular compartment. This subcellular location of PR1 is not only identified in the extracellular space but could also be in the extracellular, Vacuole, endoplasmic reticulum, endoplasmic reticulum plasma membrane, cytoplasm, and mitochondria. Figure 11. Heatmap of the subcellular localization prediction of the TdPR1 through WoLF PSORT using Tbtools v1.108. This graphic represents the prediction of subcellular localization in the different compartments (Extr: extracellular, Vacu: vacuole, Chlo: chloroplast E.R: endoplasmic reticulum, E.R. plas: endoplasmic reticulum plasma membrane, Cyto: cytoplasm, Mito: mitochondria). The intensity of color correlates with the subcellular localization prediction.

Gene Ontology (GO) Term Distribution of Triticum durum PR-1
Gene ontology (GO) analyses performed by three different servers showed variable results on biological process, molecular function, and cellular component terms for 12 TdPR1 proteins (Figure 12). Based on CELLO2GO data, three molecular functions were identified: enzyme regulator activity, ion binding, and lipid binding. The PredictProtein Figure 10. Representation of the cis-elements in the TdPR1 promoters using TBtools software v1.108. Each cis-element was identified by a unique color.

Predicting TdPR1 Pproteins Subcellular Localization
The subcellular localization of TdPR1 proteins performed by the online tool WoLF PSORT and represented by a heatmap graphic using Tbtools v1.108 are shown in Figure 11. Indeed, with a high level of prediction, 10 TdPR1 proteins were found to be located in the extracellular compartment. This subcellular location of PR1 is not only identified in the extracellular space but could also be in the extracellular, Vacuole, endoplasmic reticulum, endoplasmic reticulum plasma membrane, cytoplasm, and mitochondria.

Predicting TdPR1 Pproteins Subcellular Localization
The subcellular localization of TdPR1 proteins performed by the online tool WoLF PSORT and represented by a heatmap graphic using Tbtools v1.108 are shown in Figure  11. Indeed, with a high level of prediction, 10 TdPR1 proteins were found to be located in the extracellular compartment. This subcellular location of PR1 is not only identified in the extracellular space but could also be in the extracellular, Vacuole, endoplasmic reticulum, endoplasmic reticulum plasma membrane, cytoplasm, and mitochondria. Figure 11. Heatmap of the subcellular localization prediction of the TdPR1 through WoLF PSORT using Tbtools v1.108. This graphic represents the prediction of subcellular localization in the different compartments (Extr: extracellular, Vacu: vacuole, Chlo: chloroplast E.R: endoplasmic reticulum, E.R. plas: endoplasmic reticulum plasma membrane, Cyto: cytoplasm, Mito: mitochondria). The intensity of color correlates with the subcellular localization prediction.

Gene Ontology (GO) Term Distribution of Triticum durum PR-1
Gene ontology (GO) analyses performed by three different servers showed variable results on biological process, molecular function, and cellular component terms for 12 TdPR1 proteins (Figure 12). Based on CELLO2GO data, three molecular functions were identified: enzyme regulator activity, ion binding, and lipid binding. The PredictProtein Figure 11. Heatmap of the subcellular localization prediction of the TdPR1 through WoLF PSORT using Tbtools v1.108. This graphic represents the prediction of subcellular localization in the different compartments (Extr: extracellular, Vacu: vacuole, Chlo: chloroplast E.R: endoplasmic reticulum, E.R. plas: endoplasmic reticulum plasma membrane, Cyto: cytoplasm, Mito: mitochondria). The intensity of color correlates with the subcellular localization prediction.

Gene Ontology (GO) Term Distribution of Triticum durum PR-1
Gene ontology (GO) analyses performed by three different servers showed variable results on biological process, molecular function, and cellular component terms for 12 TdPR1 proteins ( Figure 12). Based on CELLO2GO data, three molecular functions were identified: enzyme regulator activity, ion binding, and lipid binding. The PredictProtein tool predicted that 8% of the deduced PR1 had only a chitinase activity and 11 out of 12 (91.6%) had both functions: chitinase activity and chitin binding ( Figure 12A). No result was obtained with PANNZER2. In terms of cellular components, CELLO2GO, PANNZER2, and Predict-Protein identified that TdPR1 proteins could be located in the extracellular region with a percentage of 20, 100, and 46, respectively ( Figure 12B). The biological processes regulated by all TdPR1 proteins (91.6-100%) were the response to biotic stimulus (GO:0009607) identified by the different web servers. Based on the GO enrichment analysis by PANNZER2 and PredictProtein servers, most of the PR-1 genes are implicated in defense responses (GO:0006952) (33.3%, 91.6%, respectively). Four other biological processes were found in CELLO2GO, namely transport (100%), extracellular matrix organization 100%), reproduction (66.6%), and immune system process (8.3%). Moreover, three other Gene Ontology terms were identified by the PredictProtein tool in one out of twelve TdPR1 proteins (8.3%): systemic acquired resistance (GO:0009627), response to water deprivation (GO:0009414), and response to vitamin B1 (GO:0010266) ( Figure 12C). tool predicted that 8% of the deduced PR1 had only a chitinase activity and 11 out of 12 (91.6%) had both functions: chitinase activity and chitin binding ( Figure 12A). No result was obtained with PANNZER2. In terms of cellular components, CELLO2GO, PANN-ZER2, and PredictProtein identified that TdPR1 proteins could be located in the extracellular region with a percentage of 20, 100, and 46, respectively ( Figure 12B). The biological processes regulated by all TdPR1 proteins (91.6-100%) were the response to biotic stimulus (GO:0009607) identified by the different web servers. Based on the GO enrichment analysis by PANNZER2 and PredictProtein servers, most of the PR-1 genes are implicated in defense responses (GO:0006952) (33.3%, 91.6%, respectively). Four other biological processes were found in CELLO2GO, namely transport (100%), extracellular matrix organization 100%), reproduction (66.6%), and immune system process (8.3%). Moreover, three other Gene Ontology terms were identified by the PredictProtein tool in one out of twelve TdPR1 proteins (8.3%): systemic acquired resistance (GO:0009627), response to water deprivation (GO:0009414), and response to vitamin B1 (GO:0010266) ( Figure 12C).

Differential Expression of TdPR1.2 Gene under Various Stress Conditions
The full-length cDNA sequence of TdPR1.2 (GenBank accession no. MK570869.1) was previously identified by our group [31]. To investigate the possible biological functions of the TdPR1.2 gene, we assessed the expression patterns of TdPR1.2 genes in wheat under various abiotic stress conditions using qRT-PCR (Figures 13 and 14).
In response to salt stress (150 mM NaCl), TdPR1.2 was significantly upregulated ( Figure 13A). The same result was observed when plants were subjected to mannitol and PEG stresses ( Figure 13B,C). When heat stress was applied to wheat plants (42 °C for 30 min), there was a significant increase in TdPR1.2 expression level in the roots and shoots, suggesting that this protein could have a putative protective role in controlling wheat heat tolerance ( Figure 13D).

Differential Expression of TdPR1.2 Gene under Various Stress Conditions
The full-length cDNA sequence of TdPR1.2 (GenBank accession no. MK570869.1) was previously identified by our group [31]. To investigate the possible biological functions of the TdPR1.2 gene, we assessed the expression patterns of TdPR1.2 genes in wheat under various abiotic stress conditions using qRT-PCR (Figures 13 and 14).
In response to salt stress (150 mM NaCl), TdPR1.2 was significantly upregulated ( Figure 13A). The same result was observed when plants were subjected to mannitol and PEG stresses ( Figure 13B,C). When heat stress was applied to wheat plants (42 • C for 30 min), there was a significant increase in TdPR1.2 expression level in the roots and shoots, suggesting that this protein could have a putative protective role in controlling wheat heat tolerance ( Figure 13D).
The hormonal response of TdPR1.2 gene was investigated by treating plants with salicylic acid (SA), indole acetic acid (IAA), jasmonic acid (JA), and abscisic acid (ABA). Figure 14, TdPR1.2 was upregulated in response to all hormones used in this work. Overall, these results demonstrate that TdPR1.2 is implicated in plant response to many abiotic and hormonal stresses.

Discussion
Different studies have shown that PR1 proteins play a crucial role in plants' responses to different diseases that affect common wheat. In fact, the overexpression of TaPR1-7 enhanced plant resistance to infection by Puccinia striiformis f. sp. tritici (Pst) (Stripe (yellow) rust) [53], while TcLr19PR1 [54] and TaLr35PR1 genes [55] were induced after plant infection with Puccinia triticina (leaf rust) attack. Meanwhile, TaPR1a was highly expressed and caused plant resistance to both diseases (stripe (yellow) rust and leaf rust) [56]. Previously, we have demonstrated that the newly isolated gene, TdPR1.2, presented an antibacterial and antifungal activity in vitro. Interestingly, TdPR1.2 presented a positive effect in inhibiting Septoria tritici growth in vitro [31]. Moreover, pathogenesis-related protein 1 could positively interact with other PR families. Wang et al. [57] found that TaTLP1 and TaPR1 interacted physically to protect wheat plant from leaf rust.
Furthermore, PR protein families were activated by Septoria tritici blotch disease. For instance, PR1 and PR3 were upregulated in Sevin cultivar after plant infection with Septoria tritici [58], whereas in Wangshubai, it has been demonstrated that such infection causes the upregulation of PR1 and peroxidase genes [59]. Finally, Septoria tritici infection induced the upregulation of PR-1 in Seri 82 and Frontana cultivars, respectively [60].
While different studies have investigated the role of TaPR1 proteins in plants, little is known about its homologue in durum wheat, TdPR1.2. Therefore, the genetic richness of durum wheat PR-1 genes, its molecular functions in response to abiotic and biotic stresses, is a crucial topic to understand and resolve, not only for the agronomic problems of wheat, but also for the economics and marketing of the country.
Several researchers are emphasising the identification and characterization of PR-1 proteins in different plants. They state that most PR-1s are often encoded by multi-gene families as identified in many plant species such as black pepper [19], tomato [7], and the two genomes of the banana plant [24]. In this work, we identified 12 PR-encoding genes in durum wheat genome which is less than the number of genes identified in common wheat [18], soybean [20], rice, and Arabidopsis thaliana [16] suggesting that there is no correlation between the plant genome size and the identified PR-1 members [24]. As revealed in this work, the twelve identified TdPR1 genes were distributed on five chromosomes. Two genes were present on Chr5A, Chr7A, and Chr7B, whereas only one gene was located on chromosome 6B. The rest (about a third of the selected genes) were clustered in chromosome 5B ( Figure 2, Table 1). Liu and Xue (2006) [61] explained the clustering of genes in one chromosome by the tandem gene duplication events which could occur during evolution.
A phylogenetic tree was performed by the MEGA11 software. The TdPR1 genes were subdivided into three phylogenetic groups: Group 1 (six acidic TdPR1), Group 2 (one basic TdPR1) and Group 3 (five basic TdPR1). The division of pathogenesis-related protein-1 genes into three groups based on protein isoforms (acidic/basic) have also been shown in previous studies, such as in rice [61]. The same result was found in common wheat, which has one of the biggest genomes (>16,000 megabases) among monocot agricultural plants and presented 23 PR-1 genes. The 23 TaPr-1 genes all have intron-free open reading frames that express a signal peptide at the N-terminus and a conserved PR-1-like domain, according to a sequence study of the genes. According to phylogenetic analysis, TaPr-1 genes, along with their counterparts in other monocots, form three major monophyletic groups; each group contains genes that encode basic, basic with a C-terminal extension, and acidic PR-1 proteins, respectively. This suggests that the functions of PR-1 genes in monocot plants are diverse and conserved [18]. In contrast, in sugarcane and tomato plants, pathogenesis-related proteins were clustered in the same group independently of their type (basic/acid) [7,23]. The gene structure analyses identified the presence of a single gene and a lack of intron in all TdPR1 except TdPR1.12 which presented two exons. In accordance with other plants, such as sugarcane (Saccharum spontaneum) [23], tea plant (Camellia sinensis (L.) O. Kuntze) [17], and banana (Musa balbisiana (DH-PKW)) [24], the majority of genes had only one exon and lacked introns (13 out of 19 ScPR1, 15 out of 17 CsPR1, and 10 out of 11 MaPR1, respectively). Previously, scientists have claimed that, during evolution, genes which were activated rapidly to respond to stresses were likely to decrease their intron density [62]. Zhang and his colleagues found that the expression of the CsPR1 gene, which does not have introns, were expressed more rapidly than genes with three introns in plants subjected to blight disease stress [17].
The majority of motifs are conserved among all TdPR1 proteins. The sequence alignment of the deduced protein presents two conserved domains belonging to the CRISP family. Previous studies have demonstrated that CRISPs may be involved in the plant to acquire resistance to biotic and abiotic stresses [63]. Moreover, the first isolated TdPR1.2 was aligned with eight PR-1 sequences from different species, demonstrating that the cysteine residues and the CRISP_1 domain are highly conserved in both monocotyledonous and dicotyledonous plants, whereas CRISP_2 is less conserved [11,31]. Moreover, all PR-1 proteins presented six well conserved cysteines residues [11,31,61]. These results are consistent with our findings and all the identified TdPR1 genes presented the conserved residues (Figure 1), suggesting a common molecular function of all identified PR-1 genes. In addition, the analysis of the TdPR1 gene structure and the conserved motifs indicates that TdPR1 genes are highly conserved, as demonstrated by the number of α and β helices and by the presence of signaling peptides. Such findings suggest that all the encoding proteins present the same function under environmental stress [19,24,64].
Numerous cis-elements were identified in TdPR1 genes ( Figure 10) involved in stress, development, and hormone response. Additionally, the MYB transcription factors (TFs), who had a detectable binding domain in four TdPR1 genes, have a different role, such as plant growth and development, physiological activity, primary and secondary metabolic reactions, and responses to biotic and abiotic stresses [65], suggesting the implication of these proteins in plant metabolism and development. Since our promoter regions possess a myriad of cis-elements, we suggest that the identified genes could be implicated in different molecular and metabolic pathways.
The signal peptide plays an important role in the guiding of proteins into subcellular spaces [66]. Based on the results obtained by the WoLF PSORT web server, the two proteins which do not contain the signal peptide (TdPR1.3 and TdPR1.4) are more probably localized in chloroplast and vacuole, respectively. The PR-1 gene family identified in other plants may prove our suggestion. In fact, scientists have stated that pathogenesisrelated proteins in grape and wheat containing the signal peptide could be secreted into the extracellular compartment [17,18,21]. However, in tea (Camellia sinensis (L.) O. Kuntze) and black pepper (Piper nigrum), PR-1 proteins without SPs were found in the intracellular compartment [17,19]. The acquisition of SP at the N terminal sequences might be affecting the subcellular localization of proteins. Therefore, TdPR1 proteins containing the signal peptide at the N terminal assume their guidance into the extracellular compartment in order to guarantee their cellular functions.
Putative ligand prediction shows that TdPR1.2 possess binding sites to Zn 2+ and EAH, similarly to the HbPR-1 protein from the rubber tree (Hevea brasiliensis), which had imbibed in its structure two binding sites of those ligands plus a glycerol binding domain [67]. Interestingly, no glycerol binding sites were detected in TdPR1.2 proteins.
Recently, a CaM binding domain was identified by our group and in the C-terminal part of the TdPR1.2 [31]. TdPR1.2 interacts with CaMs in a calcium-dependent manner and this interaction enhances TdPR1.2 activity, especially in the presence of Mn 2+ cations [31]. To further identify whether the other identified TdPR1 genes harbors a CaMBD in their structures, we performed in silico analysis using calmodulin target databases. In the sequence of three different PR proteins (PR1.2; PR1.3; and PR1.50), putative calmodulin-binding domains were identified. In these proteins, this domain is located at the C-terminal portion of the protein sequence. It was noted that this domain is necessary for calmodulin binding and the calcium-dependent activation of various plant proteins such as catalases [68,69], PRs [31], and mitogen-activated protein kinase phosphatases [70,71].
Calmodulins (CaMs) are omnipresent, tiny proteins with only four Ef-Hand motifs, which are common components of Ca 2+ binding proteins [68]. The main intracellular Ca 2+ signaling pathways are mediated by CaMs, and an increase in the amount of Ca 2+ in the nucleus or cytosol leads to the formation of Ca 2+ /CaM complexes, which interact with a wide range of targets, including ion transporters, protein kinases, pathogen-related proteins, transcription factors, and protein phosphatases, and control cellular functions [9,70,72,73].
It has been described in the literature that the SA and JA signaling pathways are stimulated after biotrophic/hemibiotrophic (under the control of SA) and necrotrophic (under the control of JA) pathogen infection [5]. Thus, we investigated the effect of SA and JA application on TdPR1.2 gene expression in wheat. Our results showed that TdPR1.2 was upregulated after application of those phytohormones in the roots and shoots of wheat. Other PR-1 proteins were reported to be upregulated after plant treatment with SA and JA [7,77]. In banana, MaPR1-1 was upregulated after plant treatment with SA and JA stresses due to the presence of cis-elements and binding sites for transcription factors [24]. Thus, the identification of the stress-responsive elements involved in the up/downregulation of PR-1 will help in understanding plants' resistance mechanisms toward various stresses. These findings strongly suggest that the TdPR1.2 gene plays a crucial role in plant defense against environmental stresses. It has been suggested that PR-1 genes can serve as molecular markers associated with resistance to different biotic and abiotic stresses [5,19,78]. Thus, our findings could be useful for breeding programs aimed at increasing the resistance of wheat crops to salt, drought, and hormonal stresses as well as plant infection with pathogens.

Plant Material and Stress Treatments
In this work, seeds of wheat (Triticum durum Desf.) (cv. Om Rabiaa) were sterilized in each box containing 30 mL of 0.6% NaClO solution for 15 min, then washed five times with 50 mL sterile water. For each treatment, 45 seeds were placed in each Petri dish (11 cm long, 2.5 cm high, and 11 cm wide) in the presence of a sponge and filter paper placed below to maintain moisture at 25 ± 2 • C. Seeds were then transferred to a greenhouse at 24 ± 2 • C, with photosynthetically active radiation of 280 µmol m −2 s −1 , a 16 h photoperiod, and 60 ± 10% relative humidity. After 10 days, seedlings were subjected to stresses. In this study, nine treatments were used including the control (distilled water), 150 mM NaCl, 10% PEG, 200 mM mannitol, 5 mM of each phytohormone (SA, JA, IAA, and ABA), and heat (42 • C). Each treatment was replicated three times. Finally, shoots were harvested and immediately frozen in liquid nitrogen and stored at −80 • C.

RNA Extraction and Quantitative Real-Time Reverse Transcription PCR (qRT-PCR)
Total RNA was extracted from individual roots and leaves (0.5 g of each tissue) using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Extracted RNA was then purified from genomic DNA (RNase free DNase set; QIAGEN), qualified by gel electrophoresis, and used for first-strand cDNA synthesis (GoScript Reverse Transcription System; Promega, Madison, USA) with an oligo-dT primer. PCR reactions were achieved in a 10 µL final volume tube in the presence of 3 µL cDNA (obtained from 40 ng of DNase-treated RNA), 0.5 µL of each primer of the TdPR1.2 gene at 10 µM (TdPR_Fw: 5 -ATGGCATCT TCCAAGAGT-3 and TdPR_Rv: 5 -TCA AGG GTG AGG ACG CGA A-3), 5 µL 2 × SYBR Green I master mix, and 1 µL of RNase-free water (Sigma). The reaction consisted of an initial denaturation at 95 • C for 5 min followed by 40 cycles composed of 10 s at 95 • C, 20 s at 60 • C, and 30 s at 72 • C, then a melting curve (5 s at 95 • C, 1 min at 65 • C, and 5 min with the temperature increasing from 65 to 97 • C). Three biological repetitions were performed for each experimental condition, with three technical repetitions for each sample. Melting curve analysis at the end of cycling was used to verify whether there was single amplification. At the end of the reaction, the threshold cycle (CT) values of the triplicate PCRs were averaged and used for transcript quantification. The relative expression ratio of the TdPR1.2 gene was calculated by using the comparative CT method with the actin gene designed from the T. aestivum genome (actin_Fw: 5 -TCC CTC AGC ACA TTC CAG CAGAT-3 and actin_Rv: 5 -AAC GAT TCC TGG ACC TGC CTC ATC-3 ) as an internal expression standard [113]. The relative expression level was calculated from triplicate measurements based on the 2 −∆∆CT , where ∆∆CT = (CT, target gene−CT, actin) stressed − (CT, target gene−CT, actin) control. Relative expression ratios from three independent experiments (three biological repetitions) are reported.

Statistical Analysis
Data are reported as mean ± S.E. The results were compared statistically by using Student's t test, and differences were considered significant at p < 0.01.

Conclusions
PR 1 proteins play important roles in growth regulation, development, and plant response to biotic and abiotic stress. Here, plenty of in silico tools were used to enhance our comprehensive understanding of the PR-1 family in Triticum durum plants. In fact, twelve TdPR1 were identified and clustered into three phylogenetic groups. Gene structure, conserved motifs, and physicochemical properties showed that TdPR1 proteins present highly conserved structures. Moreover, a myriad of cis-elements in the up/down stream of TdPR1 genes were found and may act in the gene expression to stress responses, hormones, and growth in durum wheat. Following cis-regulatory elements of the promoter regions, TdPR1 are clustered on chromosomes Chr5A, Chr5B, Chr6B, Chr7A, and Chr7B. All the predicted PR1 proteins identified in this work were reported to be extracellular proteins. Collectively, the findings of this study will contribute to a better understanding of molecular mechanisms and provide useful and essential information for the further functional characterization of TdPR-1 genes in future research.

Data Availability Statement:
The data generated and analyzed during this study are included in this article.