1. Introduction
Abiotic (such as flooding, drought, cold, and high temperature) and biotic (bacteria, viruses, fungi, nematodes…) stressors have an impact on plant growth and development [
1]. Those stresses are important sources of Reactive oxygen species (ROS) in cells [
1]. In many biological processes, ROS serve as signaling molecules, but an excessive buildup of ROS in living plants can cause oxidative stress damage to cells [
2,
3]. In fact, environmental constraints modify cellular redox homeostasis, inducing the over-production of small toxic compounds (ROSs), such as hydroxyl radicals (OH
−), superoxide (O
2−), hydrogen peroxide (H
2O
2), and other oxygen radicals which alter the cellular redox equilibrium [
4,
5,
6]. These extremely hazardous ROSs needs to be changed into less reactive forms in order to adapt to the living environment. To defend themselves from oxidative damage, plants have developed efficient detoxification mechanisms, including non-enzymatic and enzymatic detoxification systems [
4]. ROSs act as important second messengers in many signaling pathways, such as the increase of endogen abscisic acid (ABA) levels [
7] and different other stresses [
8,
9]. The diverse types of antioxidant enzymes found in enzymatic systems serve as the primary mechanisms for eliminating ROSs. Due to their high affinity for H
2O
2, catalases (CATs) are regarded as the most effective ROS scavengers [
5,
6]. Nearly all living organisms possess CATs, which have been extensively studied [
10]. Numerous studies have demonstrated that the regulation of plant
CAT gene expression affects how the organism develops, matures, and responds to environmental cues [
11,
12,
13]. In
Arabidopsis thaliana,
AtCAT genes encode for a small family of proteins (three isoforms known as AtCAT1, AtCAT2, and AtCAT3), which catalyze the oxidation of H
2O
2 and are crucial for maintaining the equilibrium of ROSs [
14]. The
AtCAT1 gene expression is increased by ABA through the MAPK cascades [
15,
16], but it does not appear to be affected by circadian rhythms.
AtCAT2 is mostly expressed in leaves and can be stimulated by light, cold, and possibly even the circadian rhythm [
16]. Under typical growth conditions, the
AtCAT2 mutant (
atcat2) and only 20% of the wild-type leaf catalase activity accumulate more H
2O
2 than the wild-type [
17].
AtCAT3 gene, engaged in ABA-mediated stomatal regulation in response to drought stress [
7], is controlled by CPK8 and exhibits high levels of expression throughout the entire plant at all developmental stages [
18]. Different other CAT proteins are also isolated from other plants. Three
CAT genes,
OsCATA,
OsCATB, and
OsCATC, have been discovered in rice [
9]. The most stress-responsive members of
OsCAT genes are
OsCATA and
OsCATC, according to earlier research [
9]. In durum wheat (
Triticum turgidum ssp.
durum), the first isolated
CAT gene (
TdCAT1) from the wheat genome (cv. Om Rabiaa, a local Tunisian variety) is highly expressed in the whole plant at all developmental stages [
13]. The same findings were observed in the Waha ecotype, a Saudi variety [
14], and in
Triticum monococcum [
15]. Moreover, TdCAT1 is regulated by bivalent cations and by the calcium/Calmodulin complex [
16]. Further, TdCAT1 harbors an autoinhibitory domain located at the C-terminal portion of the protein [
17]. Interestingly, no other catalase gene was identified in the durum wheat genome.
Durum wheat (
Triticum durum Desf.) is an important source of food around the globe, with an estimated 36 million tons of annual global production [
18]. Turkey and Canada are the top two producers, with an estimated 2 million ha each [
18]. India, Algeria, and Italy follow, each cultivating more than 1.5 million ha [
19,
20,
21]. Durum wheat is grown on 0.5 to 0.8 million ha annually in France, Greece, Morocco, Pakistan, Portugal, Kazakhstan, Russia, Spain, and Tunisia [
18], with approximately 0.6 million ha, Ethiopia is the largest producer of durum wheat in Sub-Saharan Africa (SSA) [
22]. This crop has a very diverse genetic heritage, and this diversity is reflected in the numerous traditional ways that it is consumed, including a number of distinctive dishes that proudly embody the national identities: pasta, couscous, bourghul, freekeh, gofio, and unleavened bread, to name a few local dishes [
22]. Durum wheat is currently grown in affluent countries primarily as a commercial crop to fuel the burgeoning food Industry, despite its close ties to traditional recipes [
22]. The most profitable crops are durum wheat and rice, with prices typically 20 to 40% higher than those of common wheat, millet, maize, and sorghum [
22]. Due to its outstanding resistance to climatic pressures, durum wheat continues to be an essential staple crop for smallholder farmers on marginal lands, but its large-scale production is closely correlated with its higher financial return.
In contrast to the research progress in other species, knowledge of the CAT genes in wheat is still limited. Therefore, in this study, a comprehensive genome-wide analysis of CAT genes in durum wheat was carried out. Indeed, phylogenetic relationships, the in silico subcellular localization, conserved domains, gene structures, gene locations, and cis-elements in the promoters of TdCAT genes were fulfilled. In addition to these latter, relative expression levels of two TdCAT genes had shown remarkable changes in response to different stress treatments, as well as plant treatment with ABA phytohormone.
3. Discussion
The catalase gene family is usually small. Genome-wide investigations of CAT families have been widely conducted as the genomes of numerous animals have been sequenced. The number of identified catalase genes in plants varies from one gene in Scots pine [
23], three genes in
Arabidopsis [
10],
N. plumbaginifolia [
24], and pumpkin [
25], four genes in rice [
12], and cucumber [
26], seven genes in
N. tabacum [
27], and cotton [
28] and ten genes in bread wheat [
29].
In order to create novel wheat cultivars with improved resistance to a variety of environmental stresses, it is necessary to understand the biological activities of
TdCAT genes and the molecular mechanisms underlying their responses to stressful situations. However, due to the intricacy of the wheat gene, the CAT family in wheat has not been adequately studied. In this study, a comprehensive genome-wide analysis of
CAT genes in durum wheat was carried out. According to their structure/functions of
CAT genes in plants, CATs are generally divided into three different groups related to vascular, photosynthetic, and reproductive functions [
24,
25,
26,
27].
In this work, six
CAT genes were identified in durum wheat genome and divided into three different classes (
Figure 1A). Among the identified genes, three genes were located on the chromosome 6B (
TdCAT3,
TdCAT4 and
TdCAT6). The
TdCAT1 and
TdCAT5 genes were mapped into chromosome 4B while
TdCAT2B was located on chromosome 6A (
Figure 2). Interestingly, through sequence alignment, results showed that the similarity of two protein sequences in each class is very high (>96%) (
Figure 3) as previously shown for CAT identified from
N. plumbaginifolia [
24]. Such result showed that
CAT genes are highly conserved during plants evolution (
Figure 2 and
Figure 3). In bread wheat, the identified genes (10 genes) were located on nine different chromosomes [
29]. The same classification was also observed in
N. plumbaginifolia [
24]. In
Nicotiana plumbaginifolia, NpCat1, NpCat2 and NpCat3 belonged to class I, class II, and class III, respectively. In
Arabidopsis, AtCAT2 belongs to class I [
30]. Similarly, our analyses showed that durum wheat CATs are also classified into 3 different classes (
Figure 1A). Moreover, phylogenetic analyses of TdCATs and CATs from other monocotyledonous and dicotyledonous species showed that all those CATs are classified into three different groups with one group harboring monocotyledonous CAT exclusively (
Figure 6).
CAT genes identified in durum wheat, as well as other
CATs identified in bread wheat,
Arabidopsis and rice were used to build a phylogenetic tree. The evolutionary tree analysis showed that TdCAT2, TdCAT3, TdCAT4 and TdCAT6 were closely related to catalase identified in bread wheat (
Figure 5). Such results suggest that CAT proteins could have specific functions. Moreover, we can suggest that the more closely related genes had a higher similarity in gene structure, but there was some variation among individual genes. In addition to the phylogenetic relationships, conserved domains, gene structures, secondary and tertiary structures, gene locations, in silico proteins localisations, cis-elements in promoters, and relative expression patterns of
TdCATs were investigated.
Gene structure has been identified as one of the representative traces of gene family evolution [
31]. Thus, the detailed structure of
TdCAT genes was analyzed to study their exon–intron organization (
Figure 2). The exons and introns of
TdCAT genes were found to be different between monocots and dicots as previously shown for bread wheat [
29]. In fact, all identified genes presented an important variation among themselves in terms of their intron/exon number and length (
Figure 1C).
All
TdCAT genes possessed two to six exons and three to five introns (
Figure 1C). The presence of large introns in TdCAT transcripts may ameliorate the recombination frequency and maintain the counterbalance of mutation bias [
12]. In bread wheat, catalase genes presented one to seven introns and two to seven exons [
29]. Moreover, a previously identified ancestral copy of a
TaCAT gene presented seven introns [
10]. Different splicing was found in
Arabidopsis family members. In fact, two kinds of splicing were found in
Arabidopsis AtCAT2, whereas four kinds of splicing were found in AtCAT3 and a total of seven proteins were encoded by
CAT genes in
Arabidopsis. In rice, OsCATA and OsCATB presented three and two kinds of variable shearing. Recently, a novel catalase gene member was found in rice genome named OsCATD. This gene encodes for a long protein of 2392 aa, the longest of all identified catalase genes Thus, this gene formed alone a clade in the phylogenetic tree (
Figure 6). Interestingly, protein sequence analysis of OsCATD showed that this protein presented an AMP-binding domain (PF00501.21), a characteristic of OsCATD that was never found in other catalase proteins [
12].
The 2D and 3D structures of the proteins were also investigated using SOPMA server. As seen in
Figure 4, the structures of all identified TdCAT proteins were predominantly formed by random coils which formed approximately half of the proteins structures followed by the alpha helixes which were concentrated on the C-terminal region of the proteins. Such results were also shown for tobacco catalase proteins [
27]. All identified catalase proteins harbor a N-glycosylation site in their structures (
Supplementary Figure S2;
Table 2). In eukaryotes, N-glycosylation is one of the most crucial protein modifications. This modification controls multiple roles in modulating plant stress tolerance. Recently, mutations in
alg3-3 and
cgl1-1, involved in N-glycosylation process causes an obvious decrease in photosynthesis in
Arabidopsis [
32]. Moreover, N-glycosylation is required for maintaining CAH1 protein stability. CAH1 is a chloroplast-located protein implicated in photosynthesis suggesting a crucial role of N-glycosylation in regulating photosynthetic efficiency. N-glycosylation is also important for protein folding and transport. In plants, it is also important for the development of stomata [
33]. Interestingly, it has been recently shown that mutation in
STT gene (
stt3a-2 mutant) causes a greater transpiration rate causing an important water loss in plants and an abnormal stomatal distribution. Thus, plants are more susceptible to drought stress [
33]. Such phenotype was related to low levels of abscisic acid and auxin in those mutants. All those phenotypes were related to an under glycosylation of AtBG1, a β-glucosidase protein controlling the transformation of conjugated IAA/ABA to active hormones confirming that N-glycosylation process is crucial for stomatal development in plants, as well as in controlling the release of active hormones to regulate plant response to abiotic stresses [
33]. In plants, the role of this conserved site in CAT plants is still unknown.
We have recently shown that durum wheat CAT (TdCAT1) harbors a conserved calmodulin binding domain located at the C-terminal portion of the protein [
16]. This interaction stimulates the catalytic activity of TdCAT1 in calcium dependent manner. Thus, identified TdCAT proteins were analyzed using Calmodulin target database server to identify the presence of Calmodulin binding domains (CaMBDs) in their structures. Our analyses showed that all identified catalases harbors at least 3 CaMBDs (
Table 4). Biological significance of those domains must be investigated in plants.
Subcellular location of proteins is an important biological characteristic of those cell components [
34]. Knowledge of proteins subcellular localizations is important to understand the mechanisms underlying protein cellular activities. Subcellular localization of different catalase proteins is already investigated. In Arabidopsis, catalase proteins are located in the main site of H
2O
2 production: peroxisomes. In rice, CAT proteins could be located in peroxisomes and cytoplasm [
12]. Moreover, TdCAT1 and TmCAT1, the first isolated CAT proteins from
T. turgidum and
T. monococcum, respectively were located into peroxisomes and the deletion of the C-terminal portion of the protein (harboring PTS1 domain) leads to the translocation of those proteins into cytosol [
15]. In bread wheat, TaCAT2A/B were localized in the cytoplasm and the nucleus [
29]. In this work, in silico analysis of the identified proteins showed that TdCAT proteins could be located into peroxisome as revealed by Pannzer and Cello2GO server whereas LocTree and Wolf PSORT suggest that those proteins could also be located into cytoplasm, chloroplast and mitochondrion (
Table 5).
Different key cellular processes (such as cell cycle control, signaling cascades, transcription regulation, and chaperone activity) are linked with disordered regions. The intrinsic flexibility of such proteins is an advantage to interact with different patterns with low affinity and high specificity [
35]. TdCAT proteins presented small, disordered regions which varies from 14.5% in TdCAT3 to 16.25% to TdCAT5 (
Supplementary Figure S3B). The presence of those regions in the TdCAT proteins suggest that those regions are related with the functions of catalase in cells and the importance of their metabolic roles in cellular regulations [
36]. In another hand, durum wheat catalase proteins have no transmembrane region in their structures (
Supplementary Figure S2).
Gene expression is controlled by the complex interaction of different cis-acting elements and trans-acting factors that participate in different pathways [
26]. Previous investigations have confirmed that catalase genes could be induced by different treatments, such as salt stress [
14,
16], cold [
10,
29,
37], drought [
7,
14,
16,
29,
37,
38], ABA [
7,
14,
16,
28,
38], SA [
16], and light [
39].
As far as we know, no research has been carried out to study the cis-acting elements of
TdCAT gene promoters. In this work, our analysis revealed the presence of different stress-responsive elements, such as anaerobic induction elements, meristem development elements, auxin responsive elements, abscissic acid responsive elements (ABRE), and light responsive, and defense and stress response (
Figure 7 and
Supplementary Figure S5). Interestingly, MYB-binding site (MBS) could be found in the promoter region of different catalase genes suggesting that some
TdCATs could be regulated by the MYB transcription factor. All those founding suggest that
TdCAT genes can be involved in plant maturation/growth and cell differentiation by acting as ROS regulators.
In the current study, we studied the transcript levels of 2 TdCATs (
TdCAT2 and
TdCAT3) genes under extreme temperatures (heat and cold), ABA, NaCl, and mannitol treatments and in three different tissues (Roots, leaves and Stems) (
Figure 8,
Figure 9,
Figure 10 and
Figure 11). Those genes belong to different subgroups and all presented a constitutive expression pattern under normal development conditions with a low expression in stems for
TdCAT2. In rice,
OsCatA and
OsCatC genes were essentially expressed in leaves in contrast to
OsCatB gene which was expressed essentially in roots [
40]. In hot peppers, the
CaCat2 gene was almost equally expressed in all tissues, whereas
CaCat1 gene was strongly expressed in vascular tissues and
CaCat3 was constitutively expressed in young seedlings and vegetative organs but with a low expression level [
41]. In addition, seven different catalase genes were isolated from
Nicotiana tabacum L., genome which were classified into 3 different groups [
27]. According to tissue-specific analyses, NtCAT1-4 was expressed strongly in the shoots, whereas NtCAT5 and NtCAT6 were expressed strongly in the roots. In another hand, the circadian rhythms influenced NtCAT7 expression. During drought stress, NtCATs expression changed the most. Moreover, during cold stress, NtCAT5, NtCAT6, and NtCAT7 expression was increased, whereas it was down-regulated under drought and salt stress [
27]. The plant’s reaction to environmental stress and H
2O
2 homeostasis in leaves is both regulated by SPCAT1 in the sweet potato (
Ipomoea batatas) [
42]. It’s interesting to note that ectopic CAT expression can influence CAT activity, as well as plant resilience to adversity. For instance, rice’s tolerance to low temperatures can be increased by the production of a wheat catalase gene [
43]. Moreover, CAT activity can be induced by the ectopic expression of maize
CAT2 (
ZmCAT2) in tobacco, enhancing pathogen resistance [
44].
TdCAT2 gene was upregulated under heat (38 °C), NaCl and mannitol but insensitive to cold stress application. Noteworthy,
TdCAT3 gene was found to be downregulated under cold stress conditions and upregulated under heat (38 °C), NaCl and mannitol suggesting that those genes could play specific roles in plants to respond to salt, drought and heat stresses. Interestingly, expression of
TdCAT2 and
TdCAT3 genes was rapidly increased when plants were subjected to ABA treatment suggesting that those catalase genes are closely related to the ABA signaling pathway in wheat. It is important to note that Gene ontology (GO) enrichment analysis showed that
TdCAT genes are largely related to ROS response, cellular organelles, antioxidant enzymes and stimulus and stress response. The same results were showed for
Brassica napus CAT [
45]. In fact,
BnCAT genes are related to stimulus responses, ROS response, and cellular organelles. Moreover, among the 14 identified genes, 10
BnCAT genes presented elevated expression levels in various tissues (roots, stem, leaf, and silique). Besides,
BnCAT1,
BnCAT2, BnCAT3,
BnCAT11,
BnCAT12 and
BnCAT13 genes were highly upregulated by salinity, cold, and two hormones (ABA, and gibberellic acid (GA)) treatment, but not by drought and methyl-jasmonate (MeJA). Nevertheless, the roles of durum wheat
TdCATs genes require further investigation. Our results open new windows for future investigations and provided insights into the
CAT family genes in durum wheat.