Next Article in Journal
LC-MS Profiling and Biological Activity of Unexplored Leucas nubica Benth. (Lamiaceae)
Previous Article in Journal
Key Genomic Regions of Rice Cultivar GuiHeFeng and Its Derivatives Revealed by Genome-Wide Analysis
Previous Article in Special Issue
Genome-Wide Identification and Expression Analysis of the WOX Family Reveals Potential Roles in Stem Development of Euphorbia hirta
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 15
Issue 4
10.3390/plants15040521
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Understanding the Role of Durum Wheat Thioredoxin h-Type TdTrxh2 in Biotic Stress Tolerance

by
Hanen Kamoun
1,
Sahar Keskes
2,
Hanen Dhouib
2,
Sana Tounsi
1,3,
Olfa Jrad
1,
Faiçal Brini
1,* and
Kaouthar Feki
1,*
1
Biotechnology and Plant Improvement Laboratory, Centre of Biotechnology of Sfax (CBS), BP1177, Sfax 3018, Tunisia
2
Laboratory of Biopesticides, Centre of Biotechnology of Sfax (CBS), BP1177, Sfax 3018, Tunisia
3
Higher School of Agriculture of Kef (ESAK), University of Jandouba, Boulifa Campus, BP7119, Kef 7100, Tunisia
*
Authors to whom correspondence should be addressed.
Plants 2026, 15(4), 521; https://doi.org/10.3390/plants15040521
Submission received: 1 January 2026 / Revised: 2 February 2026 / Accepted: 5 February 2026 / Published: 7 February 2026
(This article belongs to the Special Issue Applications of Bioinformatics in Plant Science)
Browse Figures
Versions Notes

Abstract

The thioredoxin h-type (Trxh) proteins play a crucial role as convergence points within plants’ responses to abiotic and biotic stresses. Previously, we demonstrated that the protein TdTrxh2 of durum wheat (Triticum durum Desf.) has a chaperone function and it promotes tolerance to abiotic stress. The aim of this study was to evaluate the antimicrobial effect of TdTrxh2 and its role in the response of durum wheat to Fusarium graminearum attack. First, we demonstrated the involvement of TdTrxh2 in the response of durum wheat to this fungus via the analysis of its expression profile under this fungus attack. In fact, the outcomes showed that the induction of TdTrxh2 expression is spatiotemporal in leaves and roots of durum wheat under F. graminearum infection. Interestingly, this induction was accompanied by H2O2 accumulation under short- and long-term stress in roots and leaves, respectively. Besides, the cis elements related to the two phytohormones ET and MeJA, and those implicated in defense and wound stress, were identified in the TdTrxh2 promoter’s sequence. Second, the purified TdTrxh2 protein possessed antimicrobial effects against a diverse range of bacteria and fungi in vitro. Finally, the expression of TdTrxh2 in transgenic Arabidopsis plants enhanced their tolerance to F. graminearum attack through the activation of the two H2O2-scavenging enzymes, CAT and POD, and via the induction of a subset of SA- and ABA-related genes. Moreover, the exogenous SA and ABA applications improved the growth of the transgenic lines compared to the non-transformed plants. Taken together, the results highlighted that TdTrxh2 generates tolerance of durum wheat’s response to F. graminearum attack, via the regulation of H2O2 homeostasis and the induction of hormone-associated genes. Thus, the TdTrxh2 gene could be considered as an interesting candidate gene to improve wheat tolerance to F. graminearum attack.

1. Introduction

Biotic stresses, such as attacks by pests and pathogens, disrupt various physiological and molecular mechanisms in plants, therefore affecting plant growth and productivity. Indeed, regarding the yield loss of the major food crops, wheat is about one-fifth of the global annual loss, consequently presenting a threat to global food security [1,2]. To mitigate biotic stress, plants activate various strategies at apoplastic and symplastic levels, including the activation of secondary metabolites, antioxidant enzymes, and defense proteins, like defensins, thionins, and lectins [3,4]. Under biotic stress, the recognition of pathogens via the nucleotide-binding leucine-rich repeat receptors (NLR) produces the hypersensitive response (HR), which is a local cell death. The distal parts perceive signals from the local site of stress perception and activate the systemic defense in the entire plant for subsequent attacks [5]. This rapid systemic signal is regulated in different cell compartments during stress through the reactive oxygen species (ROS), nitric oxide (NO), calcium, and hydraulic pressure [6,7]. The ROS wave is an essential signal in response not only to biotic and abiotic stresses but also in development and growth processes in plants [8]. The imbalance between ROS production and scavenging produces oxidative stress that causes widespread cellular damage. Therefore, plant cells regulate their redox environment through the activation of the ROS-scavenging enzymes, like catalase (CAT) and superoxide dismutase (SOD), the redox sensors proteins, such as peroxiredoxins (PRX), and the redox transmitter proteins, including thioredoxins (Trxs) and glutaredoxins (GRX) [9]. In the antioxidant defense mechanism, Trxs scavenge peroxides with PRX to produce water and oxygen and cause post-translational modifications, including S-nitrosylation (S-NO), S-sulfenation (S-OH), and S-thiolation (S-S), of several target proteins [10]. The regulation of several signaling proteins by S-nitrosothiols (RSNO) is a critical process in the plant immune system [11].
Trxs proteins are able to reduce disulfide bonds of target proteins through the involvement of the two cysteine residues in the WCGPC active site [11]. According to their active site, Trxs proteins are categorized as typical, featuring the canonical WCGPC motif, and atypical Trxs, possessing the WCXXC motif in the catalytic sites [12,13]. In turn, the typical Trxs are separated into several classes according to their primary structure and cellular localization. For instance, in Arabidopsis there are four chloroplastic Trxs proteins that are Trxs f-, m-, x-, and y-types, the mitochondrial Trxs o-type, and the cytosolic Trxs h-type [14]. Nevertheless, in poplar the protein Trx h-type, PtTrxh2, is present in the mitochondria, and it reduces alternative oxidase, producing its activation by pyruvate [15]. In wheat, the proteins TRXhA/B are located in the nucleus [16]. The chloroplastic Trxs f-type and the two Trx-like proteins, Trx-like 2 (TrxL2) and atypical Cys His-rich Trx (ACHT), deactivate several photosynthesis-related proteins in the dark, such as Rubisco activase and fructose-1,6-bisphosphatase, via their oxidation [17]. By contrast, depending on the light, Trxs activates these targets via the reduction of their disulfide bonds and in association with NADPH-Trx reductase C (NTRC). In addition to this Trx reductase system, Trxs are reduced in chloroplasts by the ferredoxin-dependent Trx reductase (FTR) [18]. It has been reported that the overexpression of NTRC and Trx f-type regulates the starch production in the transgenic tobacco plants [19]. So far, various functions are attributed to Trxs proteins depending on their distinct subcellular localizations and their different substrate specificities [11]. For example, in plant immunity, TRXh5 specifically reduces protein-SNO but not those regulated by S-nitrosoglutathione (GSNOR), producing an induction of the SA signaling pathway [20].
Regarding Trxs h-type, they were characterized from various plant species, and they are contributed not only in redox regulation through the reduction of disulfide bonds but also, they act as molecular chaperones, such as Trx of Arabidopsis [21,22] and TdTrxh2 of durum wheat [23]. Furthermore, Trxs h-type play a crucial role in tolerance to various abiotic stresses [24,25]. So far, the importance of Trx h-type proteins in response to biotic stress is reported in various studies. However, little is known about the role of Trx h-type in the response of durum wheat to Fusarium graminearum. The susceptibility of durum wheat to Fusarium attack poses a significant risk to its production in many regions [26,27]. Fusarium head blight (FHB), caused by Fusarium graminearum species, is a devastating disease of wheat and barley [28]. During plant–pathogen interactions, plants attribute multiple genes and activate sophisticated mechanisms to minimize the intensity of stress [29]. Among these genes, Trxh genes are crucial for plant defense against various pathogens, like LmTrxh2 of Lobularia maritima and AtTRX5 of Arabidopsis [30,31]. Despite the previous studies, the specific contribution of TdTrxh2 to the wheat–pathogen response remains poorly understood. This research highlights the efficacy of the TdTrxh2 protein as an antimicrobial agent and explores its contribution to the resistance of durum wheat against F. graminearum.

2. Results

2.1. The Induction of TdTrxh2’s Expression in Durum Wheat Under F. graminearum Infection Is Associated with H2O2 Accumulation

To evaluate the role of TdTrxh2 in the response of durum wheat to F. graminearum infection, its expression profile was examined in leaves and roots of the cv. Om Rabiaa inoculated with F. graminearum for the short and long term (6, 24, 72, and 120 h). Interestingly, the results of qRT-PCR showed that TdTrxh2 exhibits different expression patterns in these organs during stress. Indeed, during the first 6 h of inoculation, TdTrxh2 was induced, especially in roots. However, the presence of Fusarium for one day or more reprimed TdTrxh2 expression in roots. By contrast, TdTrxh2 was upregulated in leaves at different times of stress, and this induction reached its maximum after 120 h of inoculation, which was about 11-fold compared to the control condition (Figure 1A). Notably, the H2O2 levels increased significantly in roots exposed to Fusarium for 6 h, whereas in leaves the accumulation of H2O2 was observed after 24 h of inoculation. This accumulation was higher in leaves compared to root tissues exposed to Fusarium for five days (Figure 1B). Thus, these results suggested that the presence of F. graminearum induced the expression of TdTrxh2 in roots and leaves under short- and long-term stress, respectively. Furthermore, the induction of TdTrxh2 expression was accompanied by high H2O2 levels especially during long periods of stress, suggesting the involvement of this gene in response to oxidative stress produced by the Fusarium inoculation.

2.2. Analysis of the Promoter Regions of TdTrxh2 and Three Paralogous TdTrxh Genes

To understand the functions of these four TdTrxh genes in biotic stress response, the cis-regulatory elements were retrieved and classified into two groups, which are hormone-responsive elements and those related to biotic stress. This analysis showed that most TdTrxh promoters contain ABA- and MeJA-responsive elements, with the exception of TdTrxh2 and TdTrxh9 promoters, respectively. Nevertheless, the elements related to gibberellic acid (GA), which are GARE- and TGACG-motifs, were present only in the TdTrxh1 promoter region. Moreover, both TdTrxh1 and TdTrxh2 promoter regions contained elements related to ethylene (ET) response, while these elements were absent in TdTrxh3 and TdTrxh9 promoter regions. Besides, only TdTrxh3 and TdTrxh9 promoter regions harbored the TCA element that is implicated in the salicylic acid (SA) response (Figure 2). Concerning elements related to biotic stress, the WBOXNTERF3 element, which is implicated in wounding induction of the ERF3 gene [32], was detected in all these TdTrxh promoter regions, and the TdTrxh2 promoter region harbored the highest number of this element compared to the other genes. By contrast, the WUN-motif that is related to wound response [33] was absent in all these promoter regions. Furthermore, the element GT1GMSCAM4 was present in almost all these promoter regions, except for the TdTrxh2 promoter region. It has been reported previously that this element is involved in both pathogen and salt stress responses [34]. Nevertheless, TdTrxh2 and TdTrxh3 promoter regions contained TC-rich repeat, which is the cis-acting element involved in defense and stress responsiveness (Figure 2). All these in silico data may shed light on the roles of these four TdTrxh isoforms in biotic stress and phytohormone pathways.

2.3. The Antimicrobial Activity of TdTrxh2 Protein

To gain insight into the inhibitory effect of the purified His-tagged TdTrxh2 protein against various bacterial and fungal strains, several parameters were identified, including the inhibition zones of bacterial and fungal strains’ growth and the concentrations MIC, MBC, and MFC. The antibacterial activity of the His-tagged TdTrxh2 protein was assessed against Gram+ and Gram– strains and using the antibiotic Gentamicin (10 μg/well) as a positive control. The zones of inhibition for the antibiotic ranged from 14 to 25.5 mm. The data showed that the inhibition zones against Gram+ bacteria, which ranged from 16.5 to 23 mm, were higher than those against Gram– bacteria that were 12 and 13 mm (Table 1). Among the tested Gram+ bacteria, the greatest zone of inhibition was obtained against Bacillus subtilis (23 mm; Table 1; Figure 3A). Remarkably, the zones of inhibition were almost similar against the tested Gram– bacteria, about 12 to 13 mm (Table 1). On the other hand, the antibacterial activity of this protein was evaluated through the determination of the concentrations MIC and MBC. The results showed that the lowest MIC value (41 µg/mL) was obtained against B. subtilis. Moreover, the same MIC value was obtained in the case of the two Gram+ bacteria, S. aureus and B. cereus. However, the MIC value was eight-fold higher (332 µg/mL) toward the three Gram-negative strains A. tumefaciens C58 and B6 and P. aeruginosa compared to that obtained against B. subtilis (Table 2). The data showed an inverse relationship between the inhibition zone diameter and the MIC values. Indeed, the large inhibition zone observed correlates with a low MIC value, confirming the high potency of the purified protein against Gram+ strains, especially B. subtilis.
Then, MBCs were determined, and the ratios MBC/MIC were calculated in order to distinguish the inhibiting and killing ability of the purified TdTrxh2 protein. It was proposed that a compound has a bactericidal effect when the MBC/MIC ratio is less than 4, while it has bacteriostatic effect when this ratio is more than 4 [35]. Thus, TdTrxh2 exhibited bactericidal properties against S. aureus, B. subtilis, and B. cereus (Table 2). In turn, due to the inability to kill 99% of the three Gram– bacteria, the MBC values were not determined. To evaluate the antifungal activity of the purified His-tagged TdTrxh2 protein, MIC and MFC were determined against various fungi (Figure 3B). The MIC values ranged from 83 to 332 µg/mL. The lowest MIC values were obtained in the cases of F. culmorum and F. graminearum, while MIC values were 4-fold higher against A. alternata and B. cinerea (Table 3). MFC values were not determined for these fungi except for F. graminearum and B. cinerea, where the value was 332 µg/mL and the MFC/MIC ratio was not more than 4 (Table 3). Thus, the results showed that TdTrxh2 exhibited fungicidal properties against F. graminearum and B. cinerea.

2.4. Expression of TdTrxh2 Enhances Tolerance of the Transgenic Arabidopsis Lines to F. graminearum Infection

To explore the involvement of the TdTrxh2 gene in response to F. graminearum, the detached leaves of the wild-type (Wt) and the two transgenic Arabidopsis lines TH3-1 and TH6-1 were exposed to F. graminearum for five days. Interestingly, leaves of the two lines exhibited a significant resistance against this fungus compared to those of the Wt plants (Figure 4A). Indeed, a spread lesion with an area of 80 mm2 around the site of contact was observed in leaves of Wt plants, while the lesions were restricted in leaves of these two lines, about 10–30 mm2 (Figure 4B). This finding highlighted the role of TdTrxh2 in the response of durum wheat to F. graminearum attack.

2.5. Mechanism Underlying TdTrxh2’s Response to F. graminearum Infection

To gain insight on the mechanism involved in the response of transgenic Arabidopsis lines to F. graminearum, the activities of ROS-scavenging enzymes and the expression profiles of a subset of SA- and ABA-related genes were analyzed and then compared to those of the Wt plants under control and stress conditions. The outcomes demonstrated that the presence of Fusarium enhanced the activities of the antioxidant enzymes in the transgenic lines as well as in Wt plants. Nevertheless, this induction was pronounced, especially in the transgenic lines exposed to fungal infection compared to the Wt plants. Indeed, CAT and POD activities increased intensively in the two lines inoculated with Fusarium compared to the Wt plants. However, SOD activity increased not only in transgenic lines but also in Wt plants under fungal infection (Figure 4C). Thus, the expression of TdTrxh2 enhanced biotic stress tolerance of Arabidopsis plants via the activation of the two H2O2-scavenging enzymes CAT and POD. In parallel, the expression patterns of some SA- and ABA-related genes were analyzed to better understand the mechanism implicated in the TdTrxh2 gene’s response to biotic stress. As shown in Figure 5, the three genes AtPR1, AtNPR1, and AtNPR4 were upregulated in leaves of the transgenic lines TH3-1 and TH6-1 exposed to fungal infection, whereas their expression remained lower in the Wt plants. Moreover, the qRT-PCR data showed that the expression of the AtNCED3 and AtDREB2A genes that encode an enzyme in ABA biosynthesis and a transcription factor, respectively, were significantly induced in leaves of the two lines infected with Fusarium compared to the Wt plants. These results suggested that the involvement of TdTrxh2 in response to Fusarium infection could be associated with the induction of a subset of genes that are related to salicylic acid (SA) and abscisic acid (ABA) pathways.

2.6. Response of TdTrxh2-Expression Arabidopsis Lines to Phytohormone Treatments

Owing to the induction of the genes related to SA and ABA pathways in TdTrxh2-expressing Arabidopsis lines, we investigated the impact of the two phytohormones SA and ABA on the growth of the two lines TH3-1 and TH6-1. For this purpose, two-week-old seedlings of the two transgenic Arabidopsis lines and Wt plants were transferred to MS medium containing 10 µM ABA and 50 µM SA. After one week of phytohormones’ application, we observed a similar phenotype of all these seedlings under normal growth conditions. Nevertheless, the presence of SA or ABA meaningfully influenced the growth of Wt plants, whereas the two lines exhibited better growth parameters compared to Wt plants (Figure 6A). Indeed, the FW of these lines was higher than that of the Wt plants in the presence of these phytohormones, demonstrating that their aerial parts continued to grow under these conditions (Figure 6B). Moreover, SA and ABA did not inhibit the growth of the root systems of these lines, and their roots’ elongation was about 5-fold higher compared to those of the Wt plants (Figure 6C). It is worth noting that the response of these transgenic plants to SA phytohormone was better than that under ABA treatment (Figure 6). Taken together, these results suggested that TdTrxh2 confers tolerance of the transgenic lines to the exogenous application of SA and ABA phytohormones.

3. Discussion

The plants’ immune system involves a specific Trxs that possesses signaling and protective functions via the modulation of the redox status of target molecules [36]. Various studies report the role of Trxs not only in stress response but also in plant growth and development, like OsTRXh1 of rice that modulates the level of H2O2 under salt stress in OsTRXh1-overexpression plants, as well as its influence on plant development [37]. Although the role of Trxh proteins in biotic stress response is studied in various species, the function of durum wheat TdTrxh2 in response to biotic stress is still not clear until now. Thus, we aimed in this study to demonstrate the involvement of TdTrxh2 in the response to F. graminearum attack through the analysis of the transgenic TdTrxh2-expressing Arabidopsis plants and analysis of its expression in leaves and roots of durum wheat exposed to F. graminearum infection. It has been reported previously that TdTrxh2 is among the four Trxh isoforms of durum wheat that are involved in salinity tolerance via redox regulation [38]. The importance of Trx h-type in abiotic stress tolerance has been explored thoroughly in various plant species. For instance, LhTRX-h3 and AtTrx-h2 enhance drought tolerance of Liriodendron hybrid and salt tolerance of the transgenic Brassica napus plants, respectively [25,39]. On the other hand, Trxs h-type play a crucial role in defense responses during a compatible plant–pathogen interaction. In the plant–pathogen interaction, PAMPs are recognized by specific plasma membrane receptors to recognize the pathogen and initiate the PAMP-triggered immunity (PTI). This recognition is characterized by defense cascades’ reactions and induces HR [40]. In tobacco, the PAMPs molecule, flg22 from Xanthomonas axonopodis, induces ROS-scavenging enzymes and PR genes’ expression [41].
Bread wheat is more tolerant to F. pseudograminearum and F. graminearum than durum wheat, and, as a consequence, produces less yield loss [27,28]. Indeed, the strongest quantitative trait loci (QTL) associated with Fusarium crown rot (FCR) tolerance is Qcrs.cpi.3B, which is derived from the wild relative of bread wheat, Triticum spelta [42]. Moreover, in response to Fusarium infection, the differentially expressed genes are distributed on B and D subgenomes [43]. Moreover, the QTLs associated with FHB tolerance are more widely identified in bread wheat than in durum wheat [26]. In durum wheat, the QTLs associated with FHB tolerance are distributed on chromosomes 1B and 4B [44]. Moreover, Sari et al. [45] identified several QTLs associated with plant height and maturity during FHB resistance.
In the present study, we evaluated the effect of F. graminearum infection of durum wheat on TdTrxh2 expression in order to decipher its role in response to this fungus. During the response of wheat to F. graminearum infection, the majority of the genes were expressed between 48 and 72 h post-inoculation. These include encoding proteins involved in pathogen recognition, defense signaling, and resistance responses [46,47]. Our findings showed that TdTrxh2 was highly enhanced in leaves of durum wheat during 24, 72, and 120 h of inoculation. However, TdTrxh2 expression was downregulated in roots at 24 h post-inoculation (Figure 1). These data suggested that the TdTrxh2 expression level depended not only on the stage of the infection but also on the specific plant tissue. At a later stage of infection (72 h), the pathogen was colonized the subepidermal layers of the rachis, causing an extensive host cell breakdown and the emergence of arial mycelium [48]. Thus, in long-term Fusarium inoculation, it seems that an oxidative stress is produced in the aerial part of wheat, which is explored by the high H2O2 levels during 120 h of inoculation and high expression level of TdTrxh2. By contrast, during the first six hours of pathogen infection, H2O2 was exhaustively accumulated in roots with the high accumulation of TdTrxh2 transcripts (Figure 1). Interestingly, after 6 h post-inoculation, the H2O2 amount was reduced excessively in root tissues, accompanied by the downregulation of TdTrxh2. Thus, it appears that F. graminearum colonizes the durum wheat plants, reaching the aerial parts and triggering oxidative stress, as evidenced by high H2O2 accumulation and TdTrxh2 induction. Similarly, it has been reported previously that the genes PvTrxh3 and h5 of P. vulgaris are upregulated in response to symbiosis and simultaneously with H2O2 accumulation [24].
On the other hand, we examined the predicted cis elements of TdTrxh2 and then compared them to those of the three isoforms, TdTrxh1, TdTrxh3, and TdTrxh9. The outcomes showed that the TdTrxh2 promoter region contains essentially the two hormone-related stressors, which are MeJA- and ET-responsive elements, and the biotic stress-related elements, WBOXNTERF3 and TC-rich repeat elements (Figure 2). The first element is primarily implicated with wounding stress [32], while TC-rich repeat element is involved in defense and stress responsiveness. It has been reported that TC-rich repeat element is present in the promoter regions of genes involved in response to various abiotic stresses, like high temperature, salinity, and drought [49,50]. Some of these cis elements have been described in the promoter regions of Citrus sinensis CsTRXs, and CsTRXh4 harbors the highest number of regulatory elements [51]. The variability of the cis-regulatory elements in TdTrxh isoforms could be explained by their differential expression under different stresses. This is the case of TRXh genes of Arabidopsis that respond differentially to stress. For instance, the promoter region of AtTRXh5 contains many copies of the W-box (TTGACC/T) and it is induced by wounds, which is not the case of AtTRXh1, h2, h3, and h4. Furthermore, AtTRXh5 expression is enhanced by the bacteria Pseudomonas syringae via the positive regulation of the transcription factor WRKY6 [50]. There is also the AtTRXh8 gene, which is strongly induced in response to wounds, several pathogens, and elicitors [52].
Despite the importance of GT1GMSCAM4 in pathogen and salt stress responses [34], it is worth noting that this element was absent from the TdTrxh2 promoter region, suggesting the presence of other, as yet uncharacterized, regulatory elements.
Generally, the identified Trxh proteins from multiple plant species are among the components that crosstalk between abiotic and biotic stress, such as Trxh of V. vulgaris [24], A. thaliana [53], and L. maritima [30,54]. Likewise, the durum wheat TdTrxh2 plays a major role in abiotic stress tolerance, including salt, osmotic, and cadmium toxicity stresses [23]. In the second part of this study, we evaluated the antimicrobial activity of TdTrxh2 and finally the impact of its expression in transgenic Arabidopsis plants in response to F. graminearum. Our findings suggested that the TdTrxh2 protein has an effective bactericidal and fungicidal agent against the tested pathogenic strains (Figure 3). This effect could be explained by the possible interaction of TdTrxh2 with ROS-producing proteins, consequently leading to cell death. Similar to our protein, LmTrxh2 of L. maritima possesses antibacterial activity against eight pathogenic strains [30]. Likewise, AtTrxh5 and OsTrxm of Arabidopsis and O. sativa, respectively, have antifungal activity due to ROS accumulation [55,56]. Another example of plant antifungal protein is the protein OsTDX, a Trx-like protein, of O. sativa that inhibits the growth of various fungal pathogens [57]. In planta, the protective role of TdTrxh2 protein against F. graminearum was proved via the activation of the major H2O2-scavenging enzymes POD and CAT in leaves of Arabidopsis infected with this fungus (Figure 4). Likewise, the protein TdTrxh2 activates both CAT and POD in transgenic TdTrh2-expressing Arabidopsis plants in response to abiotic stress [23], suggesting the major role of TdTrxh2 in H2O2 homeostasis regulation. Furthermore, given the chaperon-like activity of TdTrxh2 [23], it is tempting to postulate that this protein stabilizes the stress-responsive proteins. Several studies highlight that Trxh genes improve tolerance of the transgenic plants to pathogen attack, such as LmTrxh2 of L. maritima that confers tolerance of the transgenic tobacco plants to multiple pathogens [30], and NtTrxh3 of Nicotiana tabacum whose overexpression enhances tolerance to tobacco mosaic virus and cucumber mosaic virus [58].
Our results suggest a potential correlation with TdTrxh2 protein and the SA and ABA signaling pathways to attenuate tolerance of the transgenic Arabidopsis plants. Previously, various studies supported the connection between Trx and phytohormone signaling pathways to control plant development and stress adaptation [59]. Some studies proved that Trxh proteins confer tolerance via the SA-associated defense signaling pathway, like bread wheat TaTrxh1 that is induced by Puccinia striiformis f. sp. tritici infection and SA treatment [60]. In pepper plants, Trx proteins function in response against Cucumber mosaic virus by promoting the expression of the defense-related genes involved in the SA pathway, which are NPR1 (non-expressor of PR gene 1), the activator of SA-mediated immune response, and PR10 (pathogenesis-related) genes [61]. Here, we demonstrated that the tolerance observed in the TdTrxh2-expressing Arabidopsis lines against F. graminearum infection is associated with the upregulation of a subset of SA-mediated genes, including NPR1, NPR4, and PR1, and the two ABA-related genes, NCED3 and DREB2A (Figure 5), suggesting a possible relation between TdTrxh2 protein and these signaling pathways. The protein NPR1 is a key node in SA signal transduction during SAR (systemic acquired resistance) response. The presence of NPR1 in the cytoplasm under SA induction is controlled by Trxs via S-nitrosylation to lead to disease resistance [62,63]. After infection, the interaction of NPR1 with NPR4 is disrupted by SA to lead to the activation of NPR1, which in turn interacts with the transcription factors TGA and WRKY to upregulate the expression of the target genes, including the antimicrobial PR genes [64,65]. In rice, OsTrxh2 and OsTrxh5 catalyze the dissociation of OsNPR1 into monomers, generating disease resistance via the activation of the OsPR1a gene. On the other hand, the protein OsTrxh2 is a direct target of XopI, which is an F-box effector of the bacteria Xanthomonas oryzae, consequently disrupting OsNPR1-mediated resistance [66]. In this study, we showed that the tolerance phenotype of the TdTrxh2-expressing Arabidopsis lines to F. graminearum infection is associated not only with the SA pathway but also the ABA signaling pathway, including NCED3 and DREB2A proteins, which are involved in ABA biosynthesis and signaling, respectively [67,68]. The inactivation of NCED3 of N. tabacum causes not only the reduction of ABA content but also affects plant growth and development, causing drought susceptibility as a consequence [69]. The phytohormone ABA interplays with other phytohormones like SA, jasmonic acid, and ethylene, contributing the HR reaction and adaptation in the long term [70,71]. Furthermore, the transcription factors are critically important in regulating diverse target genes and they are the convergence point for abiotic and biotic stress responses [72]. DREB2A regulates the expression of many drought-responsive genes in the ABA-independent pathway [73]. The ABA-independent pathway crosstalks with the ABA-dependent pathway in response to osmotic stress [74]. It is worth noting that the exogenous SA and ABA improve the growth of the transgenic TdTrxh2-expressing lines (Figure 6). Taken together, in this study, we demonstrated that TdTrxh2 plays a pivotal role in the plant defense response. However, its specific involvement in SA and ABA signaling pathways during host–pathogen interactions remains to be elucidated. Thus, further research is required to unravel the underlying mechanism of this regulation.

4. Materials and Methods

4.1. In Silico Analysis

Concerning the analysis of the promoter regions of the TdTrxh2 gene and the three genes TdTrxh1, TdTrxh3, and TdTrxh9, the promoter region of each TdTrxh gene, about 1 Kb upstream of the ATG codon, was downloaded using the EnsemblPlants server (https://plants.ensembl.org/index.html (accessed on 11 November 2024). Using the two website tools PlantCARE and PLACE [75,76], the regulatory cis elements involved in hormone and biotic stress responses were retrieved and then presented as a heatmap using TBtools software version 1.x [77]. The cis elements that were identified using the PLACE tool are WBOXNTERF3 and GT1GMSCAM4, while the others were retrieved using the PlantCARE database.

4.2. Plant Material and Stress Application

Seeds of the Tunisian durum wheat (Triticum turgidum Desf. ssp. durum), the cultivar Om Rabiaa (OR), were sterilized, as described by Tounsi et al. [78], and then placed on Petri dishes with a wet Whatman filter paper in the dark until the germination. Subsequently, they were transferred onto the Hoagland hydroponic solution in a growth room at 23 °C and 65% relative humidity, under light/dark conditions of 16 h light at 250 μmol m−2 s−1 and 8 h dark, to grow for 10 days. After that, the control plants were separated from the stressed plants that were inoculated with Fusarium graminearum strain (2 × 107 spores/mL) in the Hoagland’s solution, as detailed by Saidi et al. [79]. Before that, this strain was grown on potato dextrose agar (PDA) (Sigma-Aldrich Inc., St. Louis, MO, USA) plates for 7 days at 30 °C. After inoculation, all plants were kept in the high-humidity condition at 25 °C with a 16 h photoperiod. Leaves and root samples of the stressed and control plants were harvested at different times of stress for TdTrxh2 expression profile analysis.

4.3. RNA Isolation and qRT-PCR Analyses

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and following the protocol described by Rio et al. [80] from leaves and roots of durum wheat inoculated with F. graminearum for 6, 24, 72, and 120 h in order to perform the qRT-PCR analysis. On the other hand, to analyze the expression profiles of the genes related to SA, which were AtNPR1, AtNPR4, and AtPR1, and those related to ABA pathways, which were AtNCED3 and AtDREB2A, the total RNA from leaves of transgenic Arabidopsis TH3-1 and TH6-1 exposed or not to the fungal infection with F. graminearum was extracted using the same reagent and protocol described above. For each RNA sample, the residual genomic DNA was removed with 1 U of RNase-free DNase (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 37 °C. Then, the first-strand cDNA was synthetized using M-MLV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and the Oligo-dT primer (18 mer). The qRT-PCR preparation and reactions were performed as described by Feki et al. [81], with three technical repetitions for each sample and three biological repetitions for each experimental condition. Primers used in qRT-PCR reactions are described in Table S1. The durum wheat actin gene (TdActin) and the Arabidopsis ubiquitin 10 (UBQ10) were used as reference genes. All primers used in this analysis were obtained using Primer3web (version 4.1.0; https://primer3.ut.ee/ (accessed on 5 January 2025)) to generate amplicon sizes less than 200 pb. The relative expression of each gene was calculated using the formula 2−∆∆CT method mentioned in [82].

4.4. H2O2 Quantification

To determine the H2O2 levels in leaves and roots of durum wheat exposed to Fusarium graminearum, we followed the protocol described by Feki et al. [83].

4.5. Antibacterial Activity Tests

Previously, the His-tagged TdTrxh2 protein was expressed in the E. coli strain BL21 cells [23]. Then, its expression was induced after the addition of 1 mM IPTG (isopropyl β-d-1-thiogalactopyranoside) during 6 h at 37 °C. The phase separation with the Triton X-114 method was followed to isolate the protein without bacteria endotoxins contamination. Finally, this protein was purified using the Ni-NTA column (ThermoFisher, Inc., USA), as described by Kamoun et al. [23]. In this study, the antibacterial activity of the His-tagged TdTrxh2 protein was tested against various bacteria strains: three Gram– strains, which were Agrobacterium tumefaciens (B6: ATCC 700368), Agrobacterium tumefaciens (C58: ATCC 27072), and Pseudomonas aeruginosa (ATCC 2785), and three Gram+ strains, which were Bacillus subtilis (ATCC 6633), Bacillus cereus, and Staphylococcus aureus (ATCC 25923). These strains were obtained from a local culture collection of the Centre of Biotechnology of Sfax (CBS), Tunisia. These bacteria were cultivated in LB agar at 37 °C for 12–24 h, while Bacillus species were incubated at 30 °C. To evaluate the antibacterial activity of TdTrxh2 protein, two methods were carried out, which were the agar diffusion method [30] and the microdilution method [84]. Here, 100 µL of cell suspension was evenly spread onto the surface of LB agar. Four wells (6 mm in diameter) were carved in the agar using a sterile Pasteur pipette. Two wells were considered as positive and negative controls, containing Gentamicin (10 μg/well) and buffer solution (Tris 100 mM and NaCl 50 mM), respectively. The purified His-tagged TdTrxh2 protein was poured into the two other wells, at two different concentrations, C1 (880 µg/mL) and C2 (1660 µg/mL). Finally, the plates were incubated at 37 °C for 24 h. The antibacterial activity was assessed by measuring the diameter of the circular zones of inhibition within the well. Tests were repeated three times.
In parallel, the antibacterial test was done by the broth microdilution method in 96-well microplates to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), as described by Ben Hsouna and Hamdi [84]. To determine the MIC that corresponds to the concentration of TdTrxh2 that completely inhibited bacteria growth, 10 µL of cell suspension of different bacteria species was added to the corresponding well that contained 25 µL of thiazolyl blue tetrazolium bromide (0.5 mg/mL; (Sigma-Aldrich Inc., St. Louis, MO, USA)) and the purified TdTrxh2-His protein (1660 µg/mL). Finally, the microplates were incubated for 30 min at 37 °C. Concerning the MBC, it is defined as the lowest TdTrxh2 concentration that results in microbial death. As detailed by Ben Hsouna and Hamdi [84], MBCs were determined after the incubation of LB plates that were inoculated by 10 µL from each well, at 37 °C for 24 h.

4.6. Antifungal Activity Tests

The antifungal activity of the His-tagged TdTrxh2 protein was tested against six fungi, which were Aspergillus niger, Botrytis cinerea, Alternaria alternate, Fusarium graminearum, Fusarium oxysporum, and F. culmorum. These strains were from the Centre of Biotechnology of Sfax (CBS), Tunisia. These strains were grown on potato dextrose agar (PDA; 1.5% agar) at 28 °C for 7 days. The antifungal activity was assessed by both the well diffusion and the broth microdilution methods. The well diffusion method for the antifungal test was performed as described by Magaldi et al. [85] with modifications. The PDA medium was inoculated by different fungi (F. oxysporum, F. graminearum, and A. niger) and kept at 28 °C for 48 h. After that, three wells (6 mm in diameter) were cut out of the agar. The negative control was the buffer solution (Tris 100 mM and NaCl 50 mM). TdTrxh2 purified protein was added and the inhibition zones were observed after 24 to 72 h of incubation at 28 °C. The broth microdilution method was carried out against various phytopathogenic fungi (F. oxysporum, F. culmorum, F. graminearum, B. cinerea, A. niger, and A. alternata) to determine the minimum fungicidal concentration (MFC) of TdTrxh2, as described by Ben Hsouna and Hamdi [84]. MFC corresponds to the lowest concentration with no visible growth, with 99.5% killing of the original inoculum. These tests were performed in triplicate.

4.7. Pathogenicity Tests

To analyze the response of the transgenic Arabidopsis TH3-1 and TH6-1 lines to the F. graminearum strain, 5 µL of spore suspension (105 spores/mL in 0.001% (v/v) of Silwet L-77 solution) was deposited in the center of each detached leaf, as described by Saidi et al. [79]. Control leaves were treated only with Silwett L-77 solution, and all the detached leaves were placed in the high-humidity condition at 25 °C. After 5 days of inoculation, leaves were photographed, and the infected leaf areas were determined using the image processing and analysis program UTHSCSA (http://compdent.uthscsa.edu/dig/itdesc.html (accessed on 15 June 2025)). Experiments were performed in triplicates.

4.8. ROS-Scavenging Enzymes’ Activities

First, the proteins from the leaves (0.5 g) inoculated or not (control leaves) with F. graminearum were extracted, as described by Feki et al. [83]. Then, the protein concentration in each sample was determined according to Bradford [86]. The activities of the three ROS-scavenging enzymes CAT, SOD, and POD were determined following the protocols detailed by Feki et al. [83]. Briefly, CAT activity was determined by monitoring the disappearance of H2O2, and one unit of CAT was defined as 1 μmol/mL H2O2 decomposed per minute. SOD activity was evaluated using the photochemical NBT method. One unit of SOD was defined as the enzyme quantity required causing 50% inhibition of the rate of NBT reduction at 560 nm. POD activity was carried out by the guaiacol oxidation method. One enzyme unit of POD was defined as change in one unit of absorbance min−1.

4.9. Evaluation of Transgenic Arabidopsis Lines’ Tolerance to Hormones

The two transgenic Arabidopsis lines TH3-1 and TH6-1 expressing TdTrxh2 were obtained previously by Kamoun et al. [23]. The seeds of these two lines, and the non-transformed plants (Wt plants), were sterilized and then germinated on MS agar medium. After 15 days of growth, seedlings were transferred to MS medium containing or not 10 µM ABA and 50 µM SA and kept growing under these stress conditions for one week. The tolerance phenotype was evaluated by determining the fresh weight (FW) and the root elongation of each plant under normal and stress treatments. Each test was performed in triplicate.

4.10. Statistical Analysis

The statistical software SPSS version 20 was used for statistical analysis of the results based on the ANOVA method. Means were compared using Student’s t-test and Tukey’s HSD test. The level of significance used for all statistical tests was 5% (p < 0.05).

5. Conclusions

In summary, we shed light on the role of the durum wheat TdTrxh2 protein on biotic stress response via the analyses of expression patterns of the corresponding genes in leaves and roots of durum wheat exposed to fungal attack, its antimicrobial activity against various pathogens, and its functional characterization in the transgenic TdTrxh2-expressing Arabidopsis plants. Firstly, our results showed that F. graminearum infection led to the upregulation of TdTrxh2, with distinct temporal profiles across tissues, an immediate response in the roots, and a sustained, long-term induction in the leaves. Besides, this induction correlated closely with the accumulation of H2O2 in the affected tissues. On the other hand, the in silico analysis demonstrated the presence of elements related to ABA and MeJA hormones in wound and defense responses. Furthermore, the purified TdTrxh2 functions as a dual-action antimicrobial agent, effectively targeting both bacteria and fungi. Finally, the expression of TdTrxh2 in Arabidopsis plants conferred tolerance to F. graminearum disease via the enhancement of H2O2-scavenging enzymes’ activities and the induction of the expression of some SA- and ABA-related genes. Additionally, the application of exogenous ABA and SA improved the growth of transgenic lines. Consequently, TdTrxh2 is speculated to act as a redox regulator within the SA and ABA signaling networks, potentially mediating the plant’s transition toward pathogen tolerance. Taken together, our results suggested that TdTrxh2 could be used for engineering of crops tolerant to F. graminearum attack and, consequently, improving crop yield.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15040521/s1. Table S1: Primer details used in qRT-PCR reactions. The reference genes are UBQ10 in Arabidopsis and TdActin in wheat.

Author Contributions

Conceptualization, K.F. and F.B.; methodology, H.K. and K.F.; software, K.F.; validation, K.F. and F.B.; formal analysis, S.T. and K.F.; investigation, H.K., O.J., H.D. and S.K.; resources, F.B.; data curation, K.F. and F.B.; writing—original draft preparation, H.K. and K.F.; writing—review and editing, K.F., S.T. and F.B.; visualization, F.B. and K.F.; supervision, K.F. and F.B.; project administration, F.B.; funding acquisition, F.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Acknowledgments

This research was supported by the Ministry of Higher Education and Scientific Research, Tunisia.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Savary, S.; Willocquet, L.; Pethybridge, S.J.; Esker, P.; McRoberts, N.; Nelson, A. The global burden of pathogens and pests on major food crops. Nat. Ecol. Evol. 2019, 3, 430–439. [Google Scholar] [CrossRef] [PubMed]
  2. Fones, H.N.; Bebber, D.P.; Chaloner, T.M.; Kay, W.T.; Steinberg, G.; Gurr, S.J. Threats to global food security from emerging fungal and oomycete crop pathogens. Nat. Food 2020, 1, 332–342. [Google Scholar] [CrossRef] [PubMed]
  3. Du, B.; Haensch, R.; Alfarraj, S.; Rennenberg, H. Strategies of plants to overcome abiotic and biotic stresses. Biol. Rev. 2024, 99, 1524–1536. [Google Scholar] [CrossRef]
  4. Jain, M.; Amera, G.M.; Muthukumaran, J.; Singh, A.K. Insights into biological role of plant defense proteins: A review. Biocatal. Agric. Biotechnol. 2022, 40, 102293. [Google Scholar] [CrossRef]
  5. Fu, Z.Q.; Dong, X. Systemic acquired resistance: Turning local infection into global defense. Annu. Rev. Plant Biol. 2013, 64, 839–863. [Google Scholar] [CrossRef]
  6. Dalio, R.J.; Paschoal, D.; Arena, G.D.; Magalhaes, D.M.; Oliveira, T.S.; Merfa, M.V.; Maximo, H.J.; Machado, M.A. Hypersensitive response: From NLR pathogen recognition to cell death response. Ann. Appl. Biol. 2021, 178, 268–280. [Google Scholar] [CrossRef]
  7. Fichman, Y.; Mittler, R. Integration of electric, calcium, reactive oxygen species and hydraulic signals during rapid systemic signaling in plants. Plant J. 2021, 107, 7–20. [Google Scholar] [CrossRef]
  8. Fichman, Y.; Mittler, R. Rapid systemic signaling during abiotic and biotic stresses: Is the ROS wave master of all trades? Plant J. 2020, 102, 887–896. [Google Scholar] [CrossRef]
  9. Hasanuzzaman, M.; Bhuyan, M.H.M.B.; Zulfiqar, F.; Raza, A.; Mohsin, S.M.; Al Mahmud, J.; Fujita, M.; Fotopoulos, V. Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidant 2020, 9, 681. [Google Scholar] [CrossRef] [PubMed]
  10. Marti, M.C.; Jiménez, A.; Sevilla, F. Thioredoxin network in plant mitochondria: Cysteine S-posttranslational modifications and stress conditions. Front. Plant Sci. 2020, 11, 571288. [Google Scholar] [CrossRef]
  11. Mata-Pérez, C.; Spoel, S.H. Thioredoxin-mediated redox signalling in plant immunity. Plant Sci. 2019, 279, 27–33. [Google Scholar] [CrossRef]
  12. Dangoor, I.; Peled-Zehavi, H.; Levitan, A.; Pasand, O.; Danon, A. A small family of chloroplast atypical thioredoxins. Plant Physiol. 2009, 149, 1240–1250. [Google Scholar] [CrossRef]
  13. Chibani, K.; Wingsleb, G.; Jacquota, J.P.; Gelhayea, E.; Rouhier, N. Comparative genomic study of the thioredoxin family in photosynthetic organisms with emphasis on Populus trichocarpa. Mol. Plant 2009, 2, 308–322. [Google Scholar] [CrossRef]
  14. Meyer, Y.; Siala, W.; Bashandy, T.; Riondet, C.; Vignols, F.; Reichheld, J.P. Glutaredoxins and thioredoxins in plants. Biochim. Biophys. Acta 2008, 1783, 589–600. [Google Scholar] [CrossRef] [PubMed]
  15. Gelhaye, E.; Rouhier, N.; Gérard, J.; Jolivet, Y.; Gualberto, J.; Navrot, N.; Ohlsson, P.-I.; Wingsle, G.; Hirasawa, M.; Knaff, D.B.; et al. A specific form of thioredoxin h occurs in plant mitochondria and regulates the alternative oxidase. Proc. Natl. Acad. Sci. USA 2004, 101, 14545–14550. [Google Scholar] [CrossRef] [PubMed]
  16. Serrato, A.J.; Crespo, J.L.; Florencio, F.J.; Cejudo, F.J. Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds. Plant Mol. Biol. 2001, 46, 361–371. [Google Scholar] [CrossRef] [PubMed]
  17. Yokochi, Y.; Yoshida, K.; Hahn, F.; Miyagi, A.; Wakabayashi, K.-I.; Kawai-Yamada, M.; Weber, A.P.M.; Hisabori, T. Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment. Proc. Natl. Acad. Sci. USA 2021, 118, e2016903118. [Google Scholar] [CrossRef]
  18. Kang, Z.; Qin, T.; Zhao, Z. Thioredoxins and thioredoxin reductase in chloroplasts: A review. Gene 2019, 706, 32–42. [Google Scholar] [CrossRef]
  19. Ancín, M.; Larraya, L.; Fernández-San Millán, A.; Veramendi, J.; Burch-Smith, T.; Farran, I. NTRC and thioredoxin f overexpression differentially induces starch accumulation in tobacco leaves. Plants 2019, 8, 543. [Google Scholar] [CrossRef]
  20. Kneeshaw, S.; Gelineau, S.; Tada, Y.; Loake, G.J.; Spoel, S.H. Selective protein denitrosylation activity of Thioredoxin-h5 modulates plant Immunity. Mol. Cell 2014, 56, 153–162. [Google Scholar] [CrossRef]
  21. Lee, J.R.; Lee, S.S.; Jang, H.H.; Lee, Y.M.; Park, J.H.; Park, S.C.; Moon, J.C.; Park, S.K.; Kim, S.Y.; Lee, S.Y.; et al. Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX. Proc. Natl. Acad. Sci. USA 2009, 106, 5978–5983. [Google Scholar] [CrossRef]
  22. Park, S.K.; Jung, Y.J.; Lee, J.R.; Lee, Y.M.; Jang, H.H.; Lee, S.S.; Park, J.H.; Kim, S.Y.; Moon, J.C.; Lee, S.Y.; et al. Heat-shock and redox-dependent functional switching of an h-type Arabidopsis thioredoxin from a disulfide reductase to a molecular chaperone. Plant Physiol. 2009, 150, 552–561. [Google Scholar] [CrossRef]
  23. Kamoun, H.; Feki, K.; Tounsi, S.; Jrad, O.; Brini, F. The thioredoxin h-type TdTrxh2 protein of durum wheat confers abiotic stress tolerance of the transformant Arabidopsis plants through its protective role and the regulation of redox homoeostasis. Protoplasma 2024, 261, 317–331. [Google Scholar] [CrossRef] [PubMed]
  24. Boubakri, H.; Chihaoui, S.A.; Najjar, E.; Gargouri, M.; Barhoumi, F.; Jebara, M. Genome-wide analysis and expression profiling of H-type Trx family in Phaseolus vulgaris revealed distinctive isoforms associated with symbiotic N2-fixing performance and abiotic stress response. J. Plant Physiol. 2021, 260, 153410. [Google Scholar] [CrossRef]
  25. Tong, L.; Lin, M.; Zhu, L.; Liao, B.; Lu, L.; Lu, Y.; Chen, J.; Shi, J.; Hao, Z. Unraveling the role of the Liriodendron Thioredoxin (TRX) gene family in an abiotic stress response. Plants 2024, 13, 1674. [Google Scholar] [CrossRef]
  26. Hail, J.K.; N’Diyae, A.; Walkowiak, S.; Nilson, K.T.; Clarke, J.M.; Kutcher, H.R.; Steiner, B.; Buerstmayr, H.; Pozniak, C.J. Fusarium Head Blight in Durum Wheat: Recent Status, Breeding Directions, and Future Research Prospects. Phytopathology 2019, 109, 1664–1675. [Google Scholar] [CrossRef] [PubMed]
  27. Wachowska, U.; Wiwart, M.; Suchowilska, E.; Kwiatkowska, E.; Giedrojć, W.; Stuper-Szablewska, K.; Gontarz, D. Susceptibility of durum wheat to Fusarium graminearum, Blumeria graminis f.sp. tritici, and Zymoseptoria tritici in Poland. Sci. Rep. 2025, 15, 41235. [Google Scholar] [CrossRef]
  28. Liu, Y.; Ma, J.; Yan, G.; Zhou, M.; Wei, Y.; Zheng, Y.; Liu, C. Different Tolerance in Bread Wheat, Durum Wheat and Barley to Fusarium Crown Rot Disease Caused by Fusarium pseudograminearum. J. Phytopathol. 2012, 160, 412–417. [Google Scholar] [CrossRef]
  29. Chaurasia, S.K.; Acharya, C.; Pandey, J.K. Plant’s cellular response to biotic stress: A review. Physiol. Mol. Plant Pathol. 2025, 138, 102718. [Google Scholar] [CrossRef]
  30. Ben Saad, R.; Ben Romdhane, W.; Kukula-Koch, W.; Ben Akacha, B.; Baazaoui, N.; Bouteraa, M.T.; Chouaibi, Y.; Ben Hsouna, A.; Maisto, M.; Kačániová, M. Functional validation of Lobularia maritima thioredoxin-h2 protein for its abitlity to combat bacterial and fungal infections. Physiol. Mol. Plant Pathol. 2024, 129, 102206. [Google Scholar] [CrossRef]
  31. Sweat, T.A.; Wolpert, T.J. Thioredoxin h5 is required for victorin sensitivity mediated by a CC-NBS-LRR gene in Arabidopsis. Plant Cell 2007, 19, 673–687. [Google Scholar] [CrossRef]
  32. Nishiuchi, T.; Shinshi, H.; Suzuki, K. Rapid and Transient Activation of Transcription of the ERF3 Gene by Wounding in Tobacco Leaves. J. Biol. Chem. 2004, 279, 55355–55361. [Google Scholar] [CrossRef]
  33. Siebertz, B.; Logemann, J.; Willmitzer, L.; Schell, J. Cis-analysis of the wound-inducible promoter wun1 in transgenic tobacco plants and histochemical localization of its expression. Plant Cell 1989, 1, 961–968. [Google Scholar] [CrossRef]
  34. Park, H.C.; Kim, M.L.; Kang, Y.H.; Jeon, J.M.; Yoo, J.H.; Kim, M.C.; Park, C.Y.; Jeong, J.C.; Moon, B.C.; Lee, J.H.; et al. Pathogen- and NaCl-Induced Expression of the SCaM-4 Promoter Is Mediated in Part by a GT-1 Box That Interacts with a GT-1-Like Transcription Factor. Plant Physiol. 2004, 135, 2150–2161. [Google Scholar] [CrossRef]
  35. Pitucha, M.; Woś, M.; Miazga-Karska, M.; Klimek, K.; Mirosław, B.; Pachuta-Stec, A.; Gładysz, A.; Ginalska, G. Synthesis, antibacterial and antiproliferative potential of some new 1-pyridinecarbonyl-4-substituted thiosemicarbazide derivatives. Med. Chem. Res. 2016, 25, 1666–1677. [Google Scholar] [CrossRef]
  36. Anjou, C.; Lotoux, A.; Morvan, C.; Martin-Verstraete, I. From ubiquity to specificity: The diverse functions of bacterial thioredoxin systems. Environ. Microbiol. 2024, 26, e16668. [Google Scholar] [CrossRef] [PubMed]
  37. Zhang, C.J.; Zhao, B.C.; Ge, W.N.; Zhang, Y.F.; Song, Y.; Sun, D.Y.; Guo, Y. An apoplastic H-type thioredoxin is involved in the stress response through regulation of the apoplastic reactive oxygen species in rice. Plant Physiol. 2011, 157, 1884–1899. [Google Scholar] [CrossRef] [PubMed]
  38. Boubakri, H.; Saidi, M.N.; Barhoumi, F.; Kamoun, H.; Jebara, M.; Brini, F. Identification and characterization of Thioredoxin H-type gene family in Triticum turgidum ssp. durum in response to natural and environmental factor-induced oxidative stress. Plant Mol. Biol. Rep. 2019, 37, 464–476. [Google Scholar] [CrossRef]
  39. Ji, M.G.; Park, H.J.; Cha, J.Y.; Kim, J.A.; Shin, G.I.; Jeong, S.Y.; Lee, E.S.; Yun, D.-J.; Lee, S.Y.; Kim, W.-Y. Expression of Arabidopsis thaliana Thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance. Plant Physiol. Biochem. 2020, 147, 313–321. [Google Scholar] [CrossRef] [PubMed]
  40. Zhang, J.; Zhou, J.M. Plant immunity triggered by microbial molecular signatures. Mol. Plant 2010, 3, 783–793. [Google Scholar] [CrossRef]
  41. Mani, T.; Joshi, J.B.; Priyadharshini, R.; Sharmila, J.S.; Uthandi, S. Flagellin, a plant-defense-activating protein identified from Xanthomonas axonopodis pv. Dieffenbachiae invokes defense response in tobacco. BMC Microbiol. 2023, 23, 284. [Google Scholar] [CrossRef]
  42. Ma, J.; Li, H.B.; Zhang, C.Y.; Yang, X.M.; Liu, Y.X.; Yan, G.J.; Liu, C.J. Identification and validation of a major QTL conferring crown rot resistance in hexaploidy wheat. Theor. Appl. Genet. 2010, 120, 1119–1128. [Google Scholar] [CrossRef]
  43. Powell, J.J.; Fitzgerald, T.L.; Stiller, J.; Berkman, P.J.; Gardiner, D.M.; Manners, J.M.; Henry, R.J.; Kazan, K. The defence-associated transcriptome of hexaploid wheat displays homoeolog expression and induction bias. Plant Biotechnol. J. 2017, 15, 533–543. [Google Scholar] [CrossRef] [PubMed]
  44. Ruan, Y.; Comeau, A.; Langevin, F.; Hucl, P.; Clarke, J.M.; Brule-Babel, A.; Pozniak, C.J. Identification of novel QTL for resistance to Fusarium head blight in a tetraploid wheat population. Genome 2012, 55, 853–864. [Google Scholar] [CrossRef] [PubMed]
  45. Sari, E.; Berraies, S.; Knox, R.E.; Singh, A.K.; Ruan, Y.; Cuthbert, R.D.; Pozniak, C.J.; Henriquez, M.A.; Kumar, S.; Burt, A.J.; et al. High-density genetic mapping of Fusarium head blight resistance QTL in tetraploid wheat. PLoS ONE 2018, 13, e0204362. [Google Scholar] [CrossRef]
  46. Poland, J.A.; Balint-Kurti, P.J.; Wisser, R.J.; Pratt, R.C.; Nelson, R.J. Shades of gray: The world of quantitative disease resistance. Trends Plant Sci. 2009, 14, 21–29. [Google Scholar] [CrossRef] [PubMed]
  47. Soresi, D.; Zappacosta, D.; Garayalde, A.; Miranda, R.; Carrera, A. An in vitro assay for pre-screening resistance to Fusarium head blight in durum wheat. Phytopathol. Mediterr. 2015, 54, 253–264. [Google Scholar]
  48. Brown, N.A.; Urban, M.; van de Meene, A.M.; Hammond-Kosack, K.E. The infection biology of Fusarium graminearum: Defining the pathways of spikelet to spikelet colonisation in wheat ears. Fungal Biol. 2010, 114, 555–571. [Google Scholar] [CrossRef]
  49. Lang, H.; Jiang, Y.; Xie, Y.; Wu, J.; Wang, Y.; Jiang, M. Evolutionary and Functional Insights into Rice Universal Stress Proteins in Response to Abiotic Stresses. Biology 2025, 14, 1359. [Google Scholar] [CrossRef]
  50. Wang, C.; Han, J.; Wang, T.; Chen, C.; Liu, J.; Xu, Z.; Zhang, Q.; Wang, L.; Ren, Z. Pan-Genome-Wide dentification and Transcriptome-Wide Analysis of DREB Genes That Respond to Biotic and Abiotic Stresses in Cucumber. Agriculture 2022, 12, 1879. [Google Scholar] [CrossRef]
  51. Zuo, X.; Yang, C.; Yan, Y.; Huang, G.; Li, R. Systematic analysis of the thioredoxin gene family in Citrus sinensis: Identification, phylogenetic analysis, and gene expression patterns. Plant Signal. Behav. 2023, 18, 2294426. [Google Scholar] [CrossRef] [PubMed]
  52. Laloi, C.; Mestres-ortega, D.; Marco, Y.; Meyer, Y.; Reichheld, J. The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its W-box-mediated response to pathogen elicitor. Plant Physiol. 2004, 134, 1006–1016. [Google Scholar] [CrossRef]
  53. Belin, C.; Bashandy, T.; Cela, J.; Delorme-Hinoux, V.; Riondet, C.; Reichheld, J.P. A comprehensive study of thiol reduction gene expression under stress conditions in Arabidopsis thaliana. Plant Cell Environ. 2015, 38, 299–314. [Google Scholar] [CrossRef]
  54. Ben Saad, R.; Ben Romdhane, W.; Baazaoui, N.; Bouteraa, M.T.; Chouaibi, Y.; Mnif, W.; Ben Hsouna, A.; Kacaniova, M. Functional characterization of Lobularia maritima LmTrxh2 gene involved in cold tolerance in tobacco through alleviation of ROS damage to the plasma membrane. Int. J. Mol. Sci. 2023, 24, 3030. [Google Scholar] [CrossRef]
  55. Park, S.C.; Jung, Y.J.; Kim, I.R.; Lee, Y.; Kim, Y.-M.; Jang, M.-K.; Lee, J.R. Functional characterization of thioredoxin h type 5 with antimicrobial activity from Arabidopsis thaliana. Biotechnol. Bioproc. E 2017, 22, 129–135. [Google Scholar] [CrossRef]
  56. Park, S.C.; Kim, I.R.; Kim, J.Y.; Lee, Y.; Yoo, S.-H.; Jung, J.H.; Cheong, G.-W.; Lee, S.Y.; Jang, M.-K.; Lee, J.R. Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity. Antioxidants 2019, 8, 598. [Google Scholar] [CrossRef]
  57. Park, S.C.; Kim, I.R.; Hwang, J.E.; Kim, J.-Y.; Jung, Y.J.; Choi, W.; Lee, Y.; Jang, M.-K.; Lee, J.R. Functional Mechanisms Underlying the Antimicrobial Activity of the Oryza sativa Trx-like Protein. Int. J. Mol. Sci. 2019, 20, 1413. [Google Scholar] [CrossRef]
  58. Sun, L.; Ren, H.; Liu, R.; Li, B.; Wu, T.; Sun, F.; Liu, H.; Wang, X.; Dong, H. An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress. Mol. Plant Microbe Interact. 2010, 23, 1470–1485. [Google Scholar] [CrossRef] [PubMed]
  59. Jiménez, A.; López-Martínez, R.; Cano-Yelo, D.; Sevilla, F.; Martí, M.C. Linking redox and calcium signaling via thioredoxins. J. Exp. Bot. 2025, eraf477. [Google Scholar] [CrossRef] [PubMed]
  60. Shi, B.; Zhao, X.; Li, M.; Dong, Z.; Yang, Q.; Wang, Y.; Gao, H.; Day, B.; Ma, Q. Wheat Thioredoxin (TaTrxh1) Associates with RD19-Like cysteine protease TaCP1 to defend against stripe rust fungus through modulation of programmed cell death. Mol. Plant Microbe Interact. 2021, 34, 426–438. [Google Scholar] [CrossRef]
  61. Luna-Rivero, M.S.; Hernández-Zepeda, C.; Villanueva-Alonzo, H.; Minero-García, Y.; Castell-González, S.E.; Moreno-Valenzuela, O.A. Expression of genes involved in the salicylic acid pathway in type h1 thioredoxin transiently silenced pepper plants during a begomovirus compatible interaction. Mol. Genet. Genomics 2016, 291, 819–830. [Google Scholar] [CrossRef] [PubMed]
  62. Tada, Y.; Spoel, S.H.; Pajerowska-Mukhtar, K.; Mou, Z.; Song, J.; Wang, C.; Zuo, J.; Dong, X. Plant immunity requires conformational changes [corrected] of NPR1 via S-nitrosylation and thioredoxins. Science 2008, 321, 952–956. [Google Scholar] [CrossRef] [PubMed]
  63. Zavaliev, R.; Dong, X. NPR1, a key immune regulator for plant survival under biotic and abiotic stresses. Mol. Cell 2024, 84, 131–141. [Google Scholar] [CrossRef] [PubMed]
  64. Moreau, M.; Tian, M.; Klessig, D. Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses. Cell Res. 2012, 22, 1631–1633. [Google Scholar] [CrossRef]
  65. Ullah, C.; Chen, Y.H.; Ortega, M.A.; Tsai, C.J. The diversity of salicylic acid biosynthesis and defense signaling in plants: Knowledge gaps and future opportunities. Curr. Opin. Plant Biol. 2023, 72, 102349. [Google Scholar] [CrossRef]
  66. Ji, H.; Liu, D.; Zhang, Z.; Sun, J.; Han, B.; Li, Z. A bacterial F-box effector suppresses SAR immunity through mediating the proteasomal degradation of OsTrxh2 in rice. Plant J. 2020, 104, 1054–1072. [Google Scholar] [CrossRef]
  67. Iuchi, S.; Kobayashi, M.; Taji, T.; Naramoto, M.; Seki, M.; Kato, T.; Tabata, S.; Kakubari, Y.; Yamaguchi-Shinozaki, K.; Shinozaki, K. Regulation of drought tolerance by gene manipulation of 9-cisepoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 2001, 27, 325–333. [Google Scholar] [CrossRef]
  68. Gupta, S.; Kaur, R.; Upadhyay, A.; Chauhan, A.; Tripathi, V. Unveiling the secrets of abiotic stress tolerance in plants through molecular and hormonal insights. 3 Biotech 2024, 14, 252. [Google Scholar] [CrossRef]
  69. Yang, Y.; Zhou, Q.; Xu, J.; Li, Q.; Zhang, S. RNA interference of the NtNCDE3 reduces drought tolerance and impairs plant growth through feedback regulation of isoprenoids in Nicotiana tabacum. Environ. Exp. Bot. 2018, 155, 332–344. [Google Scholar] [CrossRef]
  70. Bharath, P.; Gahir, S.; Raghavendra, A.S. Abscisic acid-induced stomatal closure: An important component of plant defense against abiotic and biotic stress. Front. Plant Sci. 2021, 12, 615114. [Google Scholar] [CrossRef]
  71. Rai, G.K.; Khanday, D.M.; Choudhary, S.M.; Kumar, P.; Kumari, S.; Martínez-Andújar, C.; Martínez-Melgarejo, P.A.; Rai, P.K.; Pérez-Alfocea, F. Unlocking nature’s stress buster: Abscisic acid’s crucial role in defending plants against abiotic stress. Plant Stress. 2024, 11, 100359. [Google Scholar] [CrossRef]
  72. Liu, F.; Xi, M.; Liu, T.; Wu, X.; Ju, L.; Wang, D. The central role of the transcription factors in bridging biotic and abiotic stress responses for plant’s resilience. New Crops 2024, 1, 100005. [Google Scholar] [CrossRef]
  73. Singh, D.; Laxmi, A. Transcriptional regulation of drought response: A tortuous network of transcriptional factors. Front. Plant Sci. 2015, 6, 895. [Google Scholar] [CrossRef]
  74. Yoshida, T.; Mogami, J.; Yamaguchi-Shinozaki, K. ABA-dependent and ABA-independent signaling in response to osmotic stress in plants. Curr. Opin. Plant Biol. 2014, 21, 133–139. [Google Scholar] [CrossRef]
  75. Lescot, M.; Dehais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouze, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  76. Higo, K.; Ugawa, Y.; Iwamoto, M.; Korenaga, T. Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res. 1999, 27, 297–300. [Google Scholar] [CrossRef]
  77. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [Google Scholar] [CrossRef]
  78. Tounsi, S.; Kamoun, Y.; Feki, K.; Jemli, S.; Saidi, M.N.; Ziadi, H.; Alcon, C.; Brini, F. Localization and expression analysis of a novel catalase from Triticum monococcum TmCAT1 involved in response to different environmental stresses. Plant Physiol. Biochem. 2019, 139, 366–378. [Google Scholar] [CrossRef] [PubMed]
  79. Saidi, M.N.; Mergby, D.; Souibgui, A.; Yacoubi, I. Overexpression of durum wheat NAC transcription factor TtNTL3A promotes early flowering and increases multiple stress tolerance in transgenic Arabidopsis. Plant Physiol. Biochem. 2022, 192, 1–9. [Google Scholar] [CrossRef]
  80. Rio, D.C.; Ares, M., Jr.; Hannon, G.J.; Nilsen, T.W. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb. Protoc. 2010, 6, 5439. [Google Scholar] [CrossRef] [PubMed]
  81. Feki, K.; Tounsi, S.; Kamoun, H.; Al-Hashimi, A.; Brini, F. Decoding the role of durum wheat ascorbate peroxidase TdAPX7B-2 in abiotic stress response. Funct. Integr. Genomics 2024, 24, 223. [Google Scholar] [CrossRef]
  82. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  83. Feki, K.; Tounsi, S.; Brini, F. Comparison of an antioxidant system in tolerant and susceptible wheat seedlings in response to salt stress. Span. J. Agric. Res. 2017, 15, 2017. [Google Scholar] [CrossRef]
  84. Ben Hsouna, A.; Hamdi, N. Phytochemical composition and antimicrobial activities of the essential oils and organic extracts from Pelargonium graveolens growing in Tunisia. Lipids Health Dis. 2012, 11, 167. [Google Scholar] [CrossRef] [PubMed]
  85. Magaldi, S.; Mata-Essayag, S.; de Capriles, C.H.; Perez, C.; Colella, M.T.; Olaizola, C.; Ontiveros, Y. Well diffusion for antifungal susceptibility testing. Int. J. Infect. Dis. 2004, 8, 39–45. [Google Scholar] [CrossRef]
  86. Bradford, M.M. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
Plants 15 00521 g001
Figure 1. The induction of TdTrxh2 gene expression under F. graminearum infection is associated with H2O2 accumulation. (A) Expression profile of TdTrxh2 in leaves and roots of durum wheat infected by F. graminearum during 6, 24, 72, and 120 h. The expression of TdTrxh2 in leaves and roots under the control condition (without F. graminearum) was adjusted to 1. The reference gene used in the qRT-PCR reactions was TdActin. Each qRT-PCR reaction was repeated three times, and each sample was the amount of three plants per treatment. Error bars indicate standard deviation of three biological replicates. Different letters indicate significant differences (p < 0.05) between control and stressed plants. (B) Determination of H2O2 levels in leaves and roots of the inoculated durum wheat seedlings with F. graminearum during 6, 24, 72, and 120 h. Error bars indicate standard deviation of three biological replicates. Different letters in each plant tissue (leaves or roots) indicate the significant differences (p < 0.05).
Figure 1. The induction of TdTrxh2 gene expression under F. graminearum infection is associated with H2O2 accumulation. (A) Expression profile of TdTrxh2 in leaves and roots of durum wheat infected by F. graminearum during 6, 24, 72, and 120 h. The expression of TdTrxh2 in leaves and roots under the control condition (without F. graminearum) was adjusted to 1. The reference gene used in the qRT-PCR reactions was TdActin. Each qRT-PCR reaction was repeated three times, and each sample was the amount of three plants per treatment. Error bars indicate standard deviation of three biological replicates. Different letters indicate significant differences (p < 0.05) between control and stressed plants. (B) Determination of H2O2 levels in leaves and roots of the inoculated durum wheat seedlings with F. graminearum during 6, 24, 72, and 120 h. Error bars indicate standard deviation of three biological replicates. Different letters in each plant tissue (leaves or roots) indicate the significant differences (p < 0.05).
Plants 15 00521 g001
Plants 15 00521 g002
Figure 2. The cis-regulatory elements predicted in the promoter regions of TdTrxh1, TdTrxh2, TdTrxh3, and TdTrxh9. These elements are clustered in two different groups, which are hormone-responsive elements, including ABRE (ABA), CGTCA-motif (MeJA), GARE- and TGACG-motifs (GA), TCA element (SA), and ERE element (ET), and those related to biotic stress, including TC-rich repeat (cis-acting element involved in defense and stress responsiveness), GT1GMSCA (pathogen- and salt-induced gene), WBOXNTERF3 (wounding activation gene element), and WUN-motif (wound-responsive element). The figure was drawn using TBtools software version 1.x to present the putative cis elements that are spotlighted with different colors and numbers.
Figure 2. The cis-regulatory elements predicted in the promoter regions of TdTrxh1, TdTrxh2, TdTrxh3, and TdTrxh9. These elements are clustered in two different groups, which are hormone-responsive elements, including ABRE (ABA), CGTCA-motif (MeJA), GARE- and TGACG-motifs (GA), TCA element (SA), and ERE element (ET), and those related to biotic stress, including TC-rich repeat (cis-acting element involved in defense and stress responsiveness), GT1GMSCA (pathogen- and salt-induced gene), WBOXNTERF3 (wounding activation gene element), and WUN-motif (wound-responsive element). The figure was drawn using TBtools software version 1.x to present the putative cis elements that are spotlighted with different colors and numbers.
Plants 15 00521 g002
Plants 15 00521 g003
Figure 3. Growth inhibition zones of TdTrxh2 protein in different bacteria (A) and fungal (B) strains. C−: negative control. C+: Gentamicin at 10 μg/well. The purified His-tagged TdTrxh2 protein was added to two wells at different concentrations, which are C1: 880 µg/mL and C2: 1660 µg/mL. For the antifungal activity assay, experiments were done using the concentration C2.
Figure 3. Growth inhibition zones of TdTrxh2 protein in different bacteria (A) and fungal (B) strains. C−: negative control. C+: Gentamicin at 10 μg/well. The purified His-tagged TdTrxh2 protein was added to two wells at different concentrations, which are C1: 880 µg/mL and C2: 1660 µg/mL. For the antifungal activity assay, experiments were done using the concentration C2.
Plants 15 00521 g003
Plants 15 00521 g004
Figure 4. Tolerance of the two Arabidopsis lines TH3-1 and TH6-1 to Fusarium graminearum. (A) The detached leaves’ fungal test was performed and then leaves were photographed after five days of fungal infection. (B) Determination of the infected area from the Wt plants and the two lines. Asterisk indicates significant differences (p < 0.05) between transgenic lines and Wt plants. (C) Regulation of ROS-scavenging enzymes in the transgenic lines TH3-1 and TH6-1 and Wt plants infected (F: with Fusarium) or not (C: control) with F. graminearum over five days. Error bars indicate standard deviation of three biological replicates. Asterisk indicates significant differences (p < 0.05) between transgenic lines and Wt plants.
Figure 4. Tolerance of the two Arabidopsis lines TH3-1 and TH6-1 to Fusarium graminearum. (A) The detached leaves’ fungal test was performed and then leaves were photographed after five days of fungal infection. (B) Determination of the infected area from the Wt plants and the two lines. Asterisk indicates significant differences (p < 0.05) between transgenic lines and Wt plants. (C) Regulation of ROS-scavenging enzymes in the transgenic lines TH3-1 and TH6-1 and Wt plants infected (F: with Fusarium) or not (C: control) with F. graminearum over five days. Error bars indicate standard deviation of three biological replicates. Asterisk indicates significant differences (p < 0.05) between transgenic lines and Wt plants.
Plants 15 00521 g004
Plants 15 00521 g005
Figure 5. Expression profile analysis of a subset of SA- and ABA-related genes in the transgenic lines TH3-1 and TH6-1 and Wt plants infected with F. graminearum over five days. Error bars indicate standard deviation of three biological replicates. Asterisk indicates significant differences (p < 0.05).
Figure 5. Expression profile analysis of a subset of SA- and ABA-related genes in the transgenic lines TH3-1 and TH6-1 and Wt plants infected with F. graminearum over five days. Error bars indicate standard deviation of three biological replicates. Asterisk indicates significant differences (p < 0.05).
Plants 15 00521 g005
Plants 15 00521 g006
Figure 6. Response of TdTrxh2-expressing Arabidopsis plants to hormone treatments, which are 10 µM ABA and 50 µM SA. The effect of these hormones was compared to the control condition that consisted of placing two-week-old seedlings in MS medium without SA or ABA. (A) The two-week-old seedlings of the two transgenic lines (TH3-1 and TH6-1) and the non-transformed plants (Wt) were put in MS medium containing or not 10 µM ABA or 50 µM SA to grow over one week. Then, the tolerance phenotype was evaluated by measuring the fresh weight (FW) (B) and the root elongation (C) of each seedling under each stress treatment. Asterisk indicates significant differences between transgenic lines and Wt plants in each stress treatment (p < 0.05).
Figure 6. Response of TdTrxh2-expressing Arabidopsis plants to hormone treatments, which are 10 µM ABA and 50 µM SA. The effect of these hormones was compared to the control condition that consisted of placing two-week-old seedlings in MS medium without SA or ABA. (A) The two-week-old seedlings of the two transgenic lines (TH3-1 and TH6-1) and the non-transformed plants (Wt) were put in MS medium containing or not 10 µM ABA or 50 µM SA to grow over one week. Then, the tolerance phenotype was evaluated by measuring the fresh weight (FW) (B) and the root elongation (C) of each seedling under each stress treatment. Asterisk indicates significant differences between transgenic lines and Wt plants in each stress treatment (p < 0.05).
Plants 15 00521 g006
Table 1. Determination of the inhibition zone diameters (mm) of several bacteria strains using two concentrations of His-tagged TdTrxh2 protein, which are C1 (880 µg/mL) and C2 (1660 µg/mL). C+: reference antibiotic Gentamicin (10 μg/well).
Table 1. Determination of the inhibition zone diameters (mm) of several bacteria strains using two concentrations of His-tagged TdTrxh2 protein, which are C1 (880 µg/mL) and C2 (1660 µg/mL). C+: reference antibiotic Gentamicin (10 μg/well).
StrainsC1C2C+
Gram-positive
S. aureus15 ± 0.0118 ± 0.0016 ± 0.03
B. subtilis17 ± 0.0123 ± 0.0125.5 ± 0.02
B. cereus15 ± 0.0216.5 ± 0.0220.5 ± 0.02
Gram-negative
A. tumefaciens C5811 ± 0.0113 ± 0.0115 ± 0.01
A. tumefaciens B69 ± 0.0212 ± 0.0115 ± 0.01
P. aeruginosa11 ± 0.0113 ± 0.0114 ± 0.02
Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the His-tagged TdTrxh2 protein.
Table 2. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the His-tagged TdTrxh2 protein.
Bacterial StrainsMIC (µg/mL)MBC (µg/mL)MBC/MICAntibacterial
Activity
Gram-positive
S. aureus166 ± 0.01332 ± 0.052Bactericidal
B. subtilis41 ± 0.0141 ± 0.001Bactericidal
B. cereus166 ± 0.03166 ± 0.001Bactericidal
Gram-negative
A. tumefaciens C58332 ± 0.04>332- 
A. tumefaciens B6332 ± 0.03>332- 
P. aeruginosa332 ± 0.01>332- 
Table 3. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the His-tagged TdTrxh2 protein.
Table 3. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the His-tagged TdTrxh2 protein.
StrainsMIC (µg/mL)MFC (µg/mL)MFC/MICAntifungal Activity
F. culmorum83 ± 0.01>332- 
F. graminearum83 ± 0.01332 ± 0.004Fungicidal
F. oxysporum166 ± 0.02>332  
A. niger166 ± 0.01>332  
A. alternata332 ± 0.03>332  
B. cinerea332 ± 0.01332 ± 0.021Fungicidal
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Kamoun, H.; Keskes, S.; Dhouib, H.; Tounsi, S.; Jrad, O.; Brini, F.; Feki, K. Understanding the Role of Durum Wheat Thioredoxin h-Type TdTrxh2 in Biotic Stress Tolerance. Plants 2026, 15, 521. https://doi.org/10.3390/plants15040521

AMA Style

Kamoun H, Keskes S, Dhouib H, Tounsi S, Jrad O, Brini F, Feki K. Understanding the Role of Durum Wheat Thioredoxin h-Type TdTrxh2 in Biotic Stress Tolerance. Plants. 2026; 15(4):521. https://doi.org/10.3390/plants15040521

Chicago/Turabian Style

Kamoun, Hanen, Sahar Keskes, Hanen Dhouib, Sana Tounsi, Olfa Jrad, Faiçal Brini, and Kaouthar Feki. 2026. "Understanding the Role of Durum Wheat Thioredoxin h-Type TdTrxh2 in Biotic Stress Tolerance" Plants 15, no. 4: 521. https://doi.org/10.3390/plants15040521

APA Style

Kamoun, H., Keskes, S., Dhouib, H., Tounsi, S., Jrad, O., Brini, F., & Feki, K. (2026). Understanding the Role of Durum Wheat Thioredoxin h-Type TdTrxh2 in Biotic Stress Tolerance. Plants, 15(4), 521. https://doi.org/10.3390/plants15040521

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop