antioxidants

: Catalase is a crucial enzyme in antioxidant defense systems protecting eukaryotes from oxidative stress. These proteins are present in almost all living organisms and play important roles in controlling plant responses to biotic and abiotic stresses by catalyzing the decomposition of H 2 O 2 . Despite their importance, little is known about their expression in the majority of monocotyledonous species. Here, we isolated and characterized two novel catalase genes from Triticum turgidum and Hordeum vulgare , designated as TtCAT1 and HvCAT1, respectively. Phylogenetic analysis revealed that TtCAT1 and HvCAT1 presented 492 aa and shared an important identity with other catalase proteins belonging to subfamily 1. Using bioinformatic analysis, we predicted the 3D structure models of TtCAT1 and HvCAT1. Interestingly, analysis showed that the novel catalases harbor a peroxisomal targeting signal (PTS1) located at their C-terminus portion, as shown for other catalase proteins. In addition, this motif is responsible for the in silico peroxisomal localization of both proteins. Finally, RT-qPCR analysis showed that TtCAT1 and HvCAT1 are highly expressed in leaves in normal conditions but faintly in roots. Moreover, both genes are upregulated after the application of different stresses such as salt, osmotic, cold, heavy metal, and hormonal stresses. The positive responses of TtCAT1 and HvCAT1 to the various stimuli suggested that these proteins can help to protect both species against environmental stresses.


Introduction
Plants are exposed to many abiotic stresses such as water logging, drought, cold, salinity, and extreme temperatures. These stresses generate reactive oxygen species (ROS) such as superoxide oxygen (O 2 •− ) and hydrogen peroxide (H 2 O 2 ) in plant cells. ROS could be produced in different cell compartments such as chloroplasts, mitochondria, and peroxisomes [1,2]. These particles are important signaling components regulating many biological processes. Moreover, they are toxic and highly reactive at high concentrations [3,4]. In fact, ROS particles are responsible for severe damage to lipids, proteins, and nucleic acid as they impose oxidative stress. To avoid such negative effects on cells, ROS must be transformed into less dangerous forms. Plants dispose an efficient scavenging system with different antioxidant enzymes [5]. Among enzymes, catalases and peroxidases have the most efficient role due to their high affinity for H 2 O 2 to convert it to oxygen and water [6]. Catalases are powerful antioxidant metalloenzymes located in peroxisomes as shown for many other catalases such as genes isolated from Triticum durum [7] and Triticum monococcum [8]. This location is controlled by a conserved motif, a peroxisomal targeting signal type 1 (PTS1), located at the C-terminal portion of the protein, which is the most common peroxisome targeting protein in plants [9]. Animal genomes contain a single catalase gene, whereas in plants, this enzyme is decoded by a multigenic family yielding multiple isoenzymes. The number of catalases varies depending

Isolation and Molecular Cloning of TtCAT1 and HvCAT1 cDNA
Before starting, sequence alignment of different full sequences of monocotyledonous CAT1 genes was performed, and primers corresponding to the 5 and 3 UTR regions were designed to be used to amplify TtCAT1 and HvCAT1.
Total RNAs were isolated from the leaves of T. turgidum and H. vulgare plants subjected to 150 mM of NaCl for 24 h using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol. In order to eliminate the lasting genomic DNA, the extracted RNA was treated with RNase-free DNase. Then, 2 µg of total RNA were used to synthesize the first-strand cDNA using M-MLV reverse transcriptase (Invitrogen) as detailed in the manufacturer's protocol. Newly synthetized cDNAs were then used to amplify the TtCAT and HvCAT genes. After cloning and sequencing of the fragments, both full-length cDNAs were amplified using two specific primers, CAT1_Fr (5 -ATGGCATCTTCCAAGAGCAG-3 ) and CAT1_Rv (5 -TCAAGGGTGAGGACGCGAA-3 ). Specific primers were designed using the TdCAT1 sequence (accession no. KP696753) [7] and the HvCAT1 sequence (accession number XP_044983038.1). The PCR conditions were conducted as follows: an initial denaturation at Antioxidants 2022, 11, 2208 3 of 21 94 • C for 5 min followed by 30 cycles comprising a first step at 94 • C for 30 s, an annealing step at 57 • C for 30 s, an elongation step at 72 • C for 90 s, and a final 10 min extension at 72 • C. PCR products were separated by 1% agarose gel, then gel purified, cloned in the pGEM-T Easy vector, sequenced using the T7 and SP6 primers by the ABI PRISM automated sequencer, and then submitted to GenBank.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
To evaluate the expression profiles of TtCAT1 and HvCAT1, qRT-PCR was used. Thus, ten days after seed germination, stress treatments were applied. The seedlings were transferred to various solutions: 150 mM of NaCl, 150 mM of sorbitol, 150 mM of mannitol, 15% PEG (w/v), 4 • C for 4 h, heavy metals (100 µM of CdCl 2 /AlCl 3 /CuCl 2 ), and hormones (5 mM of ABA and SA). Control seedlings were kept in media without stress. Samples were collected after 24, 48, and 96 h of the stress treatments. Each tissue sample (roots and leaves) was collected and pooled together from four plants, immediately frozen in liquid nitrogen, and stored at −80 • C. For RNA extraction, we used the TRIzol method (Invitrogen) using the M-MLV reverse transcriptase for 1 h at 37 • C. The cDNA was synthetized using the oligo-dT (18mer) primer.
For expression analysis, the primers were designed using the Primer 3 software for  TtCAT1, HvCAT1, and actin genes using the following primers: qTtCAT1-F: 5 -TCTTCTCC  TACTCCGACACG-3 and qTtCAT1-R: 5 -AGGGGAAGTAGTCGACCTCC-3 ; qHvCAT1-F:  5 -ACTTGATGCTCCCCGTGAATG-3 and qHvCAT1R: 5 -GAGGCATAGGGTATTTTCA-3 ; and qAct-F: 5 -CTGACGGTGAGGACATCCAGCCCCTTG-3 and qAct-R: 5 -GCACGG CCTGAATTGCGACGTACATGG-3 . Real-time PCR (RT-PCR) was performed in 96-well plates with the CFX 96 Touch TM Real-Time PCR System (Biorad) using the SYBR@Select Master Mix for CFX (Applied Biosystems). The amplification reactions were performed in 10 µL final volumes containing 5 µL of 2 × SYBR @ Select Master Mix, 0.5 µL of each primer (10 µM), 1 µL of RNase-free water, and 3 µL of cDNA (40 ng of cDNA). The reaction consisted of an initial denaturation at 94 • C for 10 min followed by 45 cycles at 94 • C for 10 s, 60 • C for 10 s, and 72 • C for 15 s, then a melting curve (5 s at 95 • C, 1 min at 65 • C, and 5 min with temperature increasing from 65 to 97 • C). The relative expression was quantified using the comparative CT method with the actin gene as an internal expression standard [31]. The relative expression was determined using the formula 2−∆∆CT. Three biological repetitions were performed for each experimental condition, with three technical repetitions for each sample.

CAT Activity Assay
To analyze the quantitative change in catalase activity in wheat and barley after stress application for 24 h, leaves were sampled as above. For salt stress, controls were irrigated with sterile distilled water, whereas stressed plants were irrigated with 150 mM of an NaCl solution and 15% PEG (w/v). The frozen leaves of 0.2 g were homogenized in a mortar and pestle with 2.0 mL of ice-cold sodium phosphate buffer (25 mM, pH 7.0) containing 1% (w/v) soluble polyvinylpyrrolidine (PVP). The supernatant was centrifuged at 4000 g for 20 min at 4 • C. The soluble protein content was determined by the Coomassie blue dye binding method with a standard curve of bovine serum albumin [32]. After incubation at 25 • C (for blank control, incubated in boiling water for 10 min), 0.2 mL of supernatant was mixed with 1.5 mL of phosphoric acid buffer (pH 7.8) added with polyvinylpyrrolidone (PVP) and 0.3 mL of 0.1 mol/L H 2 O 2 in a 10 mL tube, which initiated the reaction. CAT activity was monitored by measuring the decrease in absorbance at 240 nm (corresponding to H 2 O 2 consumption) against a milligram of protein [33]. The decrease in absorbance was recorded by a spectrophotometer followed by the decomposition of H 2 O 2 at 240 nm and was measured for a total of 5 min [34]. One unit of enzyme activity (U) was defined as the reduced enzyme quantity per min per g of protein.
The enzyme activity was calculated as follows: AS0 means the absorbance of the blank control; and AS2 stand for the absorbance of the samples; VT means the total volume of the crude enzyme solution (mL); represents the volume of the detected crude enzyme solution (mL); FW means the fresh weight of the sample (g), and t means the time from adding H 2 O 2 to the last time (min). The activity of the catalase was calculated by the activity level of inoculation minus the level of the control at each corresponding time point.

Isolation and Sequence Analysis of the TtCAT1 and HvCAT1 Genes
The coding regions of the catalase genes from wheat and barley were amplified by PCR using the total RNA extracted from the leaves of plants of 10 days old and exposed to 150 mM of NaCl for 24 h as a template. The open reading frames of both genes are of 1476 bp nucleotides. Thus, TtCAT1 and HvCAT1 encode for precursor proteins of 492 amino acid residues as revealed by the ProtParam tool (http://web.expasy.org/protparam/; accessed on 15 May 2022), which is the same as that of other identified plant catalases such as durum wheat catalase 1 (TdCAT1, [7]), Triticum monococcum (TmCAT1, [8]), and Arabidopsis (AtCAT1, [35]). A sequence similarity search showed that HvCAT1 isolated from the Saudi Arabia variety presented 100% identity with the previously isolated sequence (accession number XP_044983038.1), whereas TtCAT1 presented 97.97% identity with TdCAT1 isolated from the Tunisian variety Om Rabiaa (Figure 1). On the other hand, TtCAT1 and TaCAT1 are related and present 100% identity ( Figure 1).
The identified catalases present an approximate molecular weight (Mw) of 56.8 kDa and a predicated isoelectric point (pI) of 6.52. In addition, the total number of negatively charged residues is almost the same (59-63 residues) in monocotyledons and dicotyledonous plants (Table 1). In addition, the GRAVY indexes of TtCAT1 and HvCAT1 are negative (−0.595 and −0.591, respectively), which means that these catalase proteins are predicted to be hydrophobic [36,37]. All studied catalase 1 proteins have a negative GRAVY index, which means that all these proteins are hydrophobic (Table 1).
A sequence similarity search showed that TtCAT1 and HvCAT1 share the highest identity with catalases isolated from monocotyledonous species such as wheat catalase TdCAT1 (Table 1). Such results suggest that TtCAT1 and HvCAT1 are similar to TmCAT1 and TdCAT1. TtCAT1 and HvCAT1 have 63 negatively charged residues (Asp + Glu) and 58 positively charged residues (Arg + Lys), which are almost the same among other isolated catalases ( Table 1). The TtCAT1 and HvCAT1 sequences were deposited into GenBank with the accession numbers of OP434464 and OP434465, respectively.
The two TtCAT1 and HvCAT1 catalytic active sites as well as heme binding motifs were detected by the ScanProsite tool. Using the same tool, 17 aa (FdReripERvvHarGAT) located at the position of 54-70 was proposed to be a catalase active site signature with the active site located at position 65 for both proteins (H residue). This domain is reported to be involved in catalytic activity, whereas a catalase proximal heme-ligand signature (RIFSYSDTQ) is composed of 9 aa and is located at the position of 344-352. This motif is involved in a direct association between the catalase and the heme group ( Figure 1; Supplemental Figure S1).
A phylogenetic relationship of TtCAT1 and HvCAT1 was constructed using the neighbour-joining method (Clustal Omega software). To make these analyses, different catalases isolated from monocots and dicotyledonous plants were used. As shown in Figure 2a, TtCAT1, isolated from the Waha variety, is closely related to Triticum aestivum (TaCAT1) and Triticum dicoccoide (TdcCAT1), which belong to class I. As shown in Figure 2a, HvCAT1 is more related to Brachypodium dictyoson BdCAT1 and the CAT1 genes isolated from dicotyledons. TtCAT1 and HvCAT1 have no predicted signal peptides at the N-terminal as revealed by SignalP-5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0; accessed on 14 May 2022; data not shown). Furthermore, the InterPro tool showed that TtCAT and HvCAT1 present a catalase core domain (IPR011614) from 18 to 401 residues and a catalase immune-responsive domain (IPR010582) from 423 to 486 ( Figure 2b). All these domains are present in monocotyledonous and dicotyledonous plants ( Figure 1). Thus, we suggested that the domain structure for catalase proteins is highly conserved in different species. Furthermore, analysis showed the conservation of the five residues that compose the heme pocket: His-65, Ser-l04, Asn-138, Arg-344, and Tyr-348 ( Figure 1). These residues form the conserved catalytic residues of the catalases [38]. These residues are conserved in many catalase proteins such as TmCAT1 [8] and TdCAT1 [7]. On the other hand, it has been recently shown that TdCAT1 harbors different cation binding domains located at different parts of the protein. The presence of 2 mM of Mn 2+ , Mg 2+ , Ca 2+ , Fe 2+ , Zn 2+ , or Cu 2+ stimu-lated the catalytic activity of TdCAT1 [39]. Sequence alignment of TtCAT1 and HvCAT1 with TdCAT1 revealed that these cation binding domains are conserved in TtCAT1 and HvCAT1 structures ( Figure 1). In fact, using the UniProt database (http://www.uniprot.org (accessed on 22 May 2022)), we performed catalase alignment with well-known Mn 2+ or Mg 2+ binding proteins. Our analysis showed that TtCAT1 harbors two conserved domains for cation binding. The first domain is located at the N-terminal portion of TtCAT1 (position 44-55; Mn 2+ binding domain), and the second one is at the C-terminal portion of the protein (position 439-449; Mg 2+ binding domain) (Figure 1 and 2b). Thus, these domains are highly conserved in different CAT1 proteins identified as previously reported [39]. The other cation motifs (calcium binding domain, copper/zinc binding domain, and iron binding domain) are also conserved ( Figure 1; Figure 2b). Using the transmembrane helix prediction (HMMTOP) database, no transmembrane helices were identified in both proteins, indicating that TtCAT1 and HvCAT1 have no function on the membranes in cells. The same result was shown for some other proteins such as PgCAT1 [40]. In addition, the carboxyl terminuses of TtCAT1 and HvCAT1 show a Q-K-L motif similar to other peroxisomal CAT proteins ( Figure 1). This motif is known to be crucial for the peroxisomal location of proteins [8]. This motif physically interacts with the receptor protein Pex5p to facilitate the penetration of catalases into peroxisomes [9,41]. On the other hand, it has been shown that the deletion of this motif causes cytoplasmic localization of TdCAT1 and TmCAT1 [8]. To confirm these hypotheses, in silico analyses of TtCAT1 and HvCAT1 using the Cello2Go

Structural Homology of TtCAT1 and HvCAT1 Proteins
Using the GenBank Blastp search, we found that TtCAT1 has the closest sequence identity to TaCAT1 and AetCAT1 with 100% and 98.98% identity, respectively. These results are consistent with the phylogenetic relationship of TtCAT1 with other monocotyledonous and dicotyledonous catalase proteins (Figure 2a). To determine the three-dimensional structure of TtCAT1 (100 % similar to HvCAT1), the PDBePISA database was used. To perform this analysis, the crystal structure of the catalase of Bacillus pumilus (pdb code: 4QOL) was used as a template [41]. This analysis shows that TtCAT1 is composed of four different subunits (which are the β-barrel domain, the N-terminal arm, the wrapping domain, and the α-helical domain) with the presence of the heme

Interaction Network of Catalase Proteins
The interaction network of the TtCAT1 and HvCAT1 proteins from wheat and barley were constructed based on the interaction relationship of the homologous CAT1 proteins from Triticum aestivum and Hordeum vulgare, respectively ( Figure 5). The interaction network analysis showed that both the TtCAT and HvCAT1 proteins interacted with acyl-coenzyme A oxidase and the FMN hydroxy acid dehydrogenase domain-containing protein (uncharacterized protein). The analysis showed that the TtCAT1 protein interacts with the PKS_ER domain-containing protein (uncharacterized protein). In barley, HvCAT1 interacts with glutathione peroxidase, the TPR_region domain-containing protein (uncharacterized protein), and Zn-Cu superoxide dismutase ( Figure 5).

RT-qPCR Analysis of TtCAT1 and HvCAT1 Gene Expressions
The tissue-specific expressions of TtCAT1 and HvCAT1 were conducted using real time qRT-PCR in different tissues of T. turgidum sp durum cv. Waha and Hordeum vulgare. Under normal conditions, TtCAT1 and HvCAT1 genes were significantly expressed in leaves, but these expressions were very low in roots of both species (Figures 6a, b). In light of these results, we suggest that TtCAT1 and HvCAT1 genes may play fundamental roles in the scavenging of H2O2 during plant development. Such findings were also shown in other plants such as Triticum monococcum [8], T. durum cv. Om Rabiaa [7], and P. ginseng [41]. The CAT1 gene was significantly expressed in N. plumbaginifolia leaves. CAT1 was

RT-qPCR Analysis of TtCAT1 and HvCAT1 Gene Expressions
The tissue-specific expressions of TtCAT1 and HvCAT1 were conducted using real time qRT-PCR in different tissues of T. turgidum sp durum cv. Waha and Hordeum vulgare. Under normal conditions, TtCAT1 and HvCAT1 genes were significantly expressed in leaves, but these expressions were very low in roots of both species (Figure 6a,b). In light of these results, we suggest that TtCAT1 and HvCAT1 genes may play fundamental roles in the scavenging of H 2 O 2 during plant development. Such findings were also shown in other plants such as Triticum monococcum [8], T. durum cv. Om Rabiaa [7], and P. ginseng [41]. The CAT1 gene was significantly expressed in N. plumbaginifolia leaves. CAT1 was also expressed with lower levels in the stem and flowers, but it was completely absent in the roots of N. plumbaginifolia [45]. In a second step, we investigated the role of TtCAT1 and HvCAT1 in plants' response to different stress conditions.
As indicated in Figure 6c, the expression of TtCAT1 is strongly induced in roots and reached its maximum accumulation after 72 h of salt stress application. This expression started to decrease after 72 h of stress application, but the expression level was higher than the normal conditions after 96 h of stress application (Figure 6c). In leaves, the expression level of TtCAT1 slightly increased and reached its maximum after 72 h of stress application with a two-fold increase in its expression level (Figure 6c). The same effect was also observed in barley as shown in Figure 6d. In the presence of PEG (15%), the catalase gene expression increased slightly in leaves with a maximum expression after 72 h of stress application, and then it started to decrease (Figure 6e), whereas in roots, the expression level of TtCAT1 increased rapidly and reached its maximum after 24 h of stress application (Figure 6e). The same result was also observed for HvCAT1 (Figure 6f).
The expressions of TtCAT1 and HvCAT1 in response to 150 mM of sorbitol and 150 mM of mannitol were the same as observed in the presence of PEG stress (Figure 7a-d). In wheat, the expression level reached its maximum after 72 h and started to decrease after 96 h. TtCAT1 and HvCAT1 genes were also expressed in response to cold stress (Figure 7ef), with maximum expressions detected after 48 h of stress application for both species (Figure 7ef).
The expression patterns of TtCAT1 and HvCAT1 were also studied in response to metallic stress. In the presence of 100 µM of CdCl 2 , the expression levels of TtCAT1 and HvCAT1 increased slightly in leaves with a maximum-fold induction after 48 h of stress application, and the expression levels started to decrease after 72 h and 96 h, respectively. In roots, the expression of these genes was strongly induced in such stress conditions with a maximum-fold induction after 72 h of stress application in both species (Figure 8a,b). The same expression pattern was observed in the presence of CuCl 2 stress with an expression level more pronounced in barley leaves compared with wheat ( Figure 8cd). Interestingly, the expression level did not decline after 96 h of stress application in both species. In presence of AlCl 3 , the expressions of TtCAT1 and HvCAT1 increased gradually (in leaves) with time and reaches their maximum after 96 h of stress application. In roots, the expression of CAT1 genes started to increase after 24 h of stress application and reaches its maximum after 72 h (Figure 8ef).
Finally, gene expression was also investigated in plants' responses to hormonal stress. As shown in Figure 9, abscisic acid induced a rapid response in the CAT1 gene in both species after 24 h of stress application and continued to increase to reach the maximum after 72 h. In wheat, this expression remained stable, whereas in barley, the expression level of HvCAT1 started to decrease (Figure 9a,b). Together, these results confirm that catalase proteins that belong to subfamily I are implicated in plant responses to different abiotic stresses.
Antioxidants 2022, 11, x FOR PEER REVIEW 13 of 21 of stress application, and then it started to decrease (Figure 6e), whereas in roots, the expression level of TtCAT1 increased rapidly and reached its maximum after 24 hours of stress application (Figure 6e). The same result was also observed for HvCAT1 (Figure 6f). The expressions of TtCAT1 and HvCAT1 in response to 150 mM of sorbitol and 150 mM of mannitol were the same as observed in the presence of PEG stress (Figures 7a-d). In wheat, the expression level reached its maximum after 72 h and started to decrease after 96 h. TtCAT1 and HvCAT1 genes were also expressed in response to cold stress (Figure 7ef), with maximum expressions detected after 48 h of stress application for both species (Figure 7ef). The expression patterns of TtCAT1 and HvCAT1 were also studied in response to metallic stress. In the presence of 100 μM of CdCl2, the expression levels of TtCAT1 and HvCAT1 increased slightly in leaves with a maximum-fold induction after 48 h of stress application, and the expression levels started to decrease after 72 h and 96 h, respectively. In roots, the expression of these genes was strongly induced in such stress conditions with a maximum-fold induction after 72 h of stress application in both species (Figure  8a,b). The same expression pattern was observed in the presence of CuCl2 stress with an ally (in leaves) with time and reaches their maximum after 96 h of stress application. In roots, the expression of CAT1 genes started to increase after 24 h of stress application and reaches its maximum after 72 h (Figure 8ef). Finally, gene expression was also investigated in plants' responses to hormonal stress. As shown in Figure 9, abscisic acid induced a rapid response in the CAT1 gene in both species after 24 h of stress application and continued to increase to reach the maximum after 72 h. In wheat, this expression remained stable, whereas in barley, the expression level of HvCAT1 started to decrease (Figure 9ab). Together, these results confirm that catalase proteins that belong to subfamily I are implicated in plant responses to different abiotic stresses.

Changes in the TtCAT and HvCAT Activities under Different Abiotic Stresses
The responses of the TtCAT1 and HvCAT1 activities to salt stress were investigated in wheat (cv. Waha) and barley, respectively. Relative to the control, both catalase protein activities increased with the duration of treatment with the tested abiotic stresses. Interestingly, the patterns varied with the type of stress ( Figure 10). Under NaCl treatment, CAT activity showed M"-type asymmetry ( Figure 10): first, it increased (at 0.5 d) and then decreased after 1 day of exposure to the stress, followed by a gradual increase from 3 days to 7 days, with the highest value (1.8-fold as the control) at day 7 (P b 0.05), and finally declined.

Changes in the TtCAT and HvCAT Activities under Different Abiotic Stresses
The responses of the TtCAT1 and HvCAT1 activities to salt stress were investigated in wheat (cv. Waha) and barley, respectively. Relative to the control, both catalase protein activities increased with the duration of treatment with the tested abiotic stresses. Interestingly, the patterns varied with the type of stress ( Figure 10). Under NaCl treatment, CAT activity showed M"-type asymmetry ( Figure 10): first, it increased (at 0.5 d) and then decreased after 1 day of exposure to the stress, followed by a gradual increase from 3 days to 7 days, with the highest value (1.8-fold as the control) at day 7 (P b 0.05), and finally declined.

Discussion
In plants, catalases are the most important scavenger proteins of H2O2. These toxic compounds could be generated during plant growth, normal metabolic processes, as well as environmental stress responses [46]. Plant catalases form a small gene family [11].

Discussion
In plants, catalases are the most important scavenger proteins of H 2 O 2 . These toxic compounds could be generated during plant growth, normal metabolic processes, as well as environmental stress responses [46]. Plant catalases form a small gene family [11]. Regarding their importance in cells, many CAT genes have been isolated and characterized in dicotyledonous and monocotyledonous species. Plant catalases have been divided into three different classes, and different genes were identified in rice [47], durum wheat cv. Om Rabiaa [7], T. monococcum [8], Triticum aestivum [13], Panax ginseng [40], Brassica napus [14], cotton [10], and cucumber plants [11]. Thus, understanding the biological roles of CAT genes in monocotyledonous and especially in durum wheat and barley as well as the molecular mechanisms regulating their responses to stresses are crucial for the identification of new wheat varieties with enhanced resistances to different environmental constraints. In the present study, we isolated the full-length catalase genes TtCAT1 from the durum wheat cv. Waha and HvCAT1 from barley cultivated in Saudi Arabia. The Waha variety is characterized by short plants resistant to many fungal diseases [48] but sensitive to salt and cold stresses. The Waha variety is cultivated in many regions of the world such as North Africa and Saudi Arabia. The phylogenetic analysis revealed that TtCAT1 is close to the TaCAT1 and HvCAT1 catalase proteins which belong to class I. In addition, bioinformatic analyses indicated that TtCAT1 harbors all the conserved domains such as the N-terminal active site (54-70) with conservation of the catalytic residue at position 65, the catalase core domain (IPR011614; position 18-401), the catalase immune-responsive domain (IPR010582; 423-486), and the catalase heme binding sites (344-352). Structure analysis showed that all these domains are identified in bread wheat, Brachypodium, and barley except for durum wheat cv. Om Rabiaa; TdCAT1 [7,8]. Thus, we can conclude that all these domains are essential for catalase activity as they are highly conserved among species. In addition, the TtCAT1 protein harbors a conserved calmodulin binding domain located at the C-terminal portion of the protein. Moreover, the 3D structure of TtCAT1 was performed using Bacillus pumilus catalase as a template [41]. This analysis showed that TtCAT1 is a tetrameric protein which is typical for other catalases such as TaCAT1 [49], TmCAT1 [8], and TdCAT1 [7]. Typical catalases contain the heme group, implicated in the catalytic activity of TtCAT1. This group is located between the internal walls of the beta barrel and several helices, with conservation of aa forming the heme group: His 65, Ser l04, Asn 138, Arg 344, and Tyr 348 [7,8,50]).
TtCAT1 harbors a putative peroxisomal localization target PTS1 in its C-terminal part. Using in silico analysis, different databases demonstrated that this domain confers a peroxisomal localization to TtCAT1. The same domain was identified in different catalases from plants such as P. ginseng [40], pumpkin catalase (Cat1) [41], T. turgidum subsp. durum cv. Om Rabiaa [7], and T. monococcum [8]. Recently, it has been demonstrated that this domain is crucial for CAT1 localization as its deletion suppresses the peroxisomal localization of TmCAT1 [8]. Interestingly, the conserved three amino acids (PSI or PSM) found in all identified catalases are not important for catalase translocation into peroxisomes [51]. It has been shown that pumpkin catalase 1 interacts with the peroxisomal biogenesis factor peroxin 5 (PEX5) to facilitate its entry into the peroxisome [51]. In addition, the leucine amino acid, located at position 11 from the C-Terminal part of AtCAT2, is crucial for protein localization as the substitution of the leucine residue by a glycine inhibits the catalase import to peroxisome [52]. This localization may suggest that catalase could play a crucial role in photorespiration, hormone metabolism, lipid metabolism, and plant responses to surrounding stresses [53].
During recent years, S-nitrosylation has been considered as the most investigated protein post-translational modification (PTM). S-nitrosylation controls many processes such as hormone signaling in plants via a group of proteins called transnitrosylase [54]. In plants, Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are controlled by S-nitrosylation (regulated via catalase proteins; [54]). In Arabidopsis, it has been shown that AtCAT3 has a transnitrosylase activity at Cys-10, and it is required for S-nitrosylation to regulate oxide signaling in Arabidopsis via the highly conserved residue Cyc-343 present in AtCAT3 and not AtCAt2 proteins [55]. In this work, we showed that TtCAT1 and HvCAT1 presented four and five nitrosylated sites, respectively (Supplemental Figure S3). C86, C230, C325, and C370 residues were conserved in both proteins, whereas HvCAT presented another site at C421. These results suggest that further investigations are needed to establish where TtCAT1 and HvCAT1 can be nitrosylated in wheat and barley, respectively. Mutations of these residues are needed to clarify the role of these residues in vivo during plant responses to different stimuli as well as during hormonal signaling and plant development. In fact, different studies revealed that S-nitrosylation is detected during plant responses to auxin signaling via the TIR1 protein in A. thaliana and Triticum aestivum L. [6,56]. BIN2 isolated from Zea mays was also shown to positively regulate brassinosteroid signaling in plants [6].
The presence of nitrosylated sites in catalase proteins was shown in bovine catalase, which presented one site at Cys 377 in the LGPNYLQIPVNCPYR motif and at Cys370 in sweet pepper (Capsicum annuum) [57]. Four cysteine residues were detected in the durum wheat (cv. Om Rabiaa) TdCAT1 sequence. The C86, C230, and C325 residues were conserved, but TdCAT1 presented a putative S-nitrosylated residue at the C470 position absent in TtCAT1. The three C86, C230, and C325 residues are also conserved in different monocotyledonous and dicotyledonous catalases [44].
In different plants, it has been shown that catalase genes play crucial roles in controlling plants' responses to biotic and abiotic stresses [6][7][8]18,40]. In the Waha variety, we have no information about the molecular regulation during plants' responses to abiotic stresses. In the Om Rabiaa variety, researchers demonstrated that TdCAT1 was upregulated by salt stress (with maximum induction after 3 days), PEG induced osmotic stress (with maximum induction after 2 days), H 2 O 2 (oxidative stress with maximum induction after 24 h), and manganese (maximum induction after 6 days) [7]. In order to investigate the role of TtCAT1 in Triticum turgidum var Waha, its expression profile was analyzed in different tissues and under various environmental conditions. Under standard conditions, TtCAT1 is highly expressed in leaves (Figure 6a) as previously shown for other plants such as AtCAT2 in Arabidopsis [18], PgCAT1 in Panax ginseng [40], TdCAT1 in Triticum turgidum subsp durum cv. Om Rabiaa [7], and TmCAT1 in Triticum monococcum [8]. The same effect was observed in barley, where HvCAT1 was slightly induced in roots but highly induced in leaves under normal conditions (Figure 6b). Interestingly, stress application enhances TtCAT1 expression with differential expression patterns between organs. The same effect was also reported for TmCAT1 [8].

Conclusions
In conclusion, two peroxisomal catalase encoding genes (TtCAT1 from durum wheat and HvCAT1 from barley) were identified and cloned. TtCAT and HvCAT1 proteins are 492 aa in length and have a predicted molecular weight of 56.8 kDa. Moreover, the presence of the conserved domains, such as the catalytic active site, the peroxisomal targeting sequence, the calmodulin binding domain, and the heme binding motif, confirmed that it is a putative peroxisomal catalase. Using qRT-PCR analysis, we showed that TtCAT1 and HvCAT1 genes were expressed relatively high in leaves and stems comparing with roots in standard conditions. TtCAT1 and HvCAT1 were induced rapidly in 10-day-old seedlings using various stimuli such as osmotic agents (mannitol, PEG, and NaCl treatment), heavy metals (copper and cadmium treatments), and chilling stress. The positive responses of TtCAT1 and HvCAT1 to different abiotic stresses suggest that these catalases may play crucial roles in stress responses.