Pyroxsulam Boosts Wheat Tolerance to Bixlozone: Effects and Mechanisms

Abstract

Bixlozone has significant potential as a pre-emergence and early post-emergence herbicide in wheat production across China. However, its safety when applied to wheat is poor, frequently causing phytotoxicity. This study confirmed the capacity of pyroxsulam to mitigate bixlozone-induced injury without compromising weed control efficacy and examined the underlying processes. Wheat exposed to bixlozone alone developed pronounced chlorosis and significant reductions in tiller formation, ultimately lowering the grain yield. In contrast, co-treatment with pyroxsulam substantially relieved chlorosis and restored both the tiller number and yield performance. Bixlozone reduced chlorophyll and carotenoid levels, impairing the function of photosystem II (PSII), which was mitigated by pyroxsulam application. Transcriptome profiling showed that pyroxsulam strongly activated detoxification-related pathways, especially those associated with glutathione S-transferases (GSTs) and ATP-binding cassette (ABC) transporters, which facilitate the metabolic detoxification or compartmentalization of bixlozone. qRT-PCR further validated the marked induction of key detoxification genes ABCC8-X1, GSTU6 and ABCC10 following pyroxsulam addition. Collectively, this study provides initial mechanistic insight into how pyroxsulam, though itself a herbicide, can enhance wheat tolerance to bixlozone by stimulating endogenous detoxification systems. These findings offer a framework for the development of safer herbicide mixtures and expand current understanding of crop–herbicide interactions, providing meaningful implications for herbicide management and sustainable agricultural systems.

1. Introduction

Wheat (Triticum aestivum L.) is a cornerstone of global food production and remains essential for sustaining food security [1]. Weed interference represents a major biotic stressor that constrains both the yield and grain quality, and it is a particularly serious challenge for ensuring stable wheat production in China. Because of their effectiveness and relatively low cost, herbicides have become integral to modern wheat-growing systems and are widely used to suppress weed pressure [2].

Alopecurus myosuroides is an aggressive, highly tillering grass weed that is capable of thriving under diverse environmental conditions. Increasing infestation levels can lead to severe yield losses, estimated between 4.6% and 62.3% [3]. Over the past decade, repeated use of herbicides such as clodinafop-propargyl and mesosulfuron-methyl has selected for resistance in many A. myosuroides populations, further complicating effective chemical control [4,5,6,7].

Unlike conventional herbicides, bixlozone exhibits a novel mode of action, providing effective control against resistant A. myosuroides. This makes bixlozone an important herbicide with profound application value for wheat production in China. Bixlozone is a deoxy-D-ribulose-5-phosphate synthase (DXS) inhibitor. After uptake, it is transported through the xylem and interferes with diterpene biosynthesis, disrupting photosynthesis and causing rapid necrosis in susceptible weeds. However, the application of bixlozone can induce considerable phytotoxic stress in wheat, seen in significant (over 90%) leaf chlorosis, plant wilting, and reductions of 90.41% and 62.06% in fresh weight and height, respectively, after the application of 400 mg/L bixlozone [8]. In severe cases, it leads to plant withering and death, resulting in significant yield losses. This not only restricts the popularization and application of this herbicide, but also poses potential risks to the safe production of wheat. Therefore, exploring the occurrence mechanism of its phytotoxicity and corresponding mitigation strategies is of great practical significance for wheat production.

For exploring methods to mitigate the phytotoxicity of bixlozone to crops, one possible solution is to enhance the metabolic capacity of wheat itself. Plant detoxification of xenobiotic compounds, including herbicides, typically involves three major stages [9]: (1) primary metabolism by cytochrome P450 monooxygenases (CYP450s), which introduce polar functional groups via oxidation, reduction, or hydrolysis to enhance solubility [10,11]; (2) conjugation reactions mediated by glutathione S-transferases (GSTs), which attach glutathione (GSH) to herbicides or their metabolites, generating less toxic or nontoxic conjugates [12]; and (3) transport processes facilitated by ATP-binding cassette (ABC) transporters, which compartmentalize or export herbicides and their conjugates to reduce their concentrations at the biochemical target site [13,14]. Activation of these detoxification pathways can enable plants to effectively reduce the concentration and toxicity of absorbed exogenous compounds.

Numerous previous studies have confirmed that herbicide safeners can regulate the above-mentioned detoxification pathways to improve crop tolerance to herbicides. Safeners, such as mefenpyr-diethyl, isoxadifen-ethyl and cloquintocet-mexyl, can specifically induce the expression of CYP450 family genes (e.g., CYP71, CYP76) in wheat, enhance the activity of GSTs and the content of GSH, and up-regulate the expression of ABC transporters, thereby synergistically promoting the phase I oxidation, phase II conjugation and phase III compartmentalization transport of herbicides, and reducing herbicide toxicity [15,16,17]. For example, cloquintocet-mexyl can significantly accelerate the metabolism rate of pyroxsulam in wheat and improve wheat’s tolerance to pyroxsulam [17].

Herbicide–herbicide interactions can be categorized into three types: synergism, additive effect, and antagonism. Among these, synergism has been more extensively studied, while research reports on herbicide–herbicide antagonism are relatively scarce. Li et al. found that 2,4-D exhibited significant antagonism against glyphosate in glyphosate-resistant Echinochloa colona populations, whereas only a weak antagonistic effect was observed in sensitive populations. This antagonism was associated with a reduced uptake of glyphosate and, to a lesser extent, impaired translocation [18]. Wu et al. found that bixlozone mixed with diflufenican or pyroxasulfone exhibited significant synergistic effects, while the mixture of bixlozone and cyanazine showed obvious antagonistic effects [19]. Furthermore, reported cases of antagonism between herbicides have focused primarily on the antagonistic effects concerning weed control efficacy; antagonistic effects on crops have not yet been reported.

Interestingly, when bixlozone is applied together with the ALS-inhibiting sulfonylurea herbicide pyroxsulam, the severity of bixlozone-induced damage is markedly reduced. This apparent “detoxification” suggests that pyroxsulam may stimulate endogenous protective pathways or detoxification systems in the wheat, thereby enhancing tolerance to bixlozone.

Based on the information outlined above, we propose several hypotheses. First, pyroxsulam may induce a stress response that triggers a broad detoxification program in wheat, thereby helping plants to maintain normal growth under chemical stress. Second, multiple herbicides may converge on shared defense-signaling networks, suggesting that pyroxsulam could function as an elicitor that specifically activates transcription of core detoxification genes or enhances their protein accumulation. Third, this induction may accelerate the metabolism of bixlozone through pathways such as cytochrome P450-mediated hydroxylation, GST-driven glutathione conjugation, and ABC transporter-mediated sequestration or efflux. These processes would decrease the concentration of active bixlozone at its target site, substantially reducing the phytotoxic symptoms.

The objective of this study was therefore to integrate physiological analyses, transcriptomic sequencing, and molecular verification to determine whether pyroxsulam modulates bixlozone metabolism in wheat by selectively inducing detoxification-related gene expression, ultimately reducing or preventing herbicide injury. The results clarify the molecular basis of the “detoxification effect” observed when both herbicides are co-applied and provide a theoretical foundation for designing herbicide combinations that are characterized by improved safety and efficiency. The findings contribute to a broader understanding of detoxification regulatory networks that protect plants from xenobiotic stress. Additionally, this study proposes a novel strategy for enhancing the selectivity of bixlozone between wheat and A. myosuroides, which is beneficial for the promotion and application of bixlozone and for safeguarding wheat production security.

2. Results and Discussion

2.1. Pyroxsulam Did Not Reduce Bixlozone’s Efficacy Against Alopecurus aequalis

At the standard field concentration (B-F vs. MIX-F), the plant control efficacies of bixlozone against A. aequalis were 91.62% (B-F) and 96.92% (MIX-F), while the fresh weight control efficacies were 93.10% (B-F) and98.28% (MIX-F). At the 75% standard concentration (B-F_l vs. MIX-F_l), the plant control efficacies were 81.22% (B-F_l) and 92.41% (MIX-F_l), with fresh weight control efficacies of 82.18% (B-F_l) and 93.68% (MIX-F_l) (Figure 1a,b). The control efficacies of mixed treatment were higher than those of bixlozone (p < 0.05). The interaction between bixlozone and pyroxsulam was determined to be additive. These results indicate that adding pyroxsulam to bixlozone does not inhibit, but rather maintains or slightly improves, the control efficacy against the target weed A. aequalis.

2.2. Pyroxsulam Mitigates Bixlozone-Associated Phytotoxicity

2.2.1. Effects on Symptoms of Phytotoxicity in Wheat

Field observations revealed that wheat plants exposed to bixlozone exhibited pronounced chlorosis and bleaching, reaching a severity rating of grade 3, in sharp contrast to the control and other treatments (Figure 2a,c). Collectively, these observations confirm the physiological basis of bixlozone toxicity in wheat. Importantly, when bixlozone was applied together with pyroxsulam, the phytotoxicity rating decreased to grade 1, and chlorosis was notably reduced, although mild discoloration remained (Figure 2b,d).

Laboratory toxicity assays and the phenotypes in Figure 2 corroborated the field observations. No symptoms of plant damage were visible the day after herbicide treatment. However, on days 2 and 4 following treatment, plants treated solely with bixlozone developed marked chlorosis and bleaching, reaching injury of grade 3. In contrast, pyroxsulam alone caused negligible damage and did not impede growth (Figure 3), confirming its high selectivity toward wheat [20]. The mixture treatment significantly reduced the injury severity to grade 1, aligning with trends observed in the field. Together, these results indicate that pyroxsulam can effectively attenuate the phytotoxic effects of bixlozone.

Regarding the observation of herbicide injury grades, it is necessary to clarify that the reported injury grades in this study are a descriptive presentation. A statistical analysis of the experimental data, such as plant height, yield, and chlorophyll content, will be provided in the following sections to quantitatively describe the effect of pyroxsulam in alleviating bixlozone-induced injury in wheat.

In addition, commercial formulations were used for the field test while high-purity technical material was used for the laboratory test. The core objective of the field experiment was to simulate the actual application conditions in agricultural production. The commercial formulation contains legally compliant adjuvants such as surfactants and solvents, which can restore the actual spreading, penetration, efficacy and phytotoxicity performance of the herbicide under field conditions. This ensures that the research results are instructive for practical production and conforms to the general specifications of field efficacy trials. The primary purpose of the laboratory experiment was to accurately dissect the physiological responses, metabolic pathways and herbicide interaction mechanisms in wheat mediated by the active ingredient itself, rather than formulation adjuvants. Using high-purity technical material can eliminate the interference of non-target variables such as surfactants and solvents in commercial formulations, ensuring that the experimental results specifically reflect the biological effects of the active ingredient and improving the accuracy and reliability of the mechanistic analysis. These two experimental systems are independent and complementary, serving for practical production verification and molecular and physiological mechanistic dissection, respectively, with no mutual interference between the results.

2.2.2. Effects on Wheat Tillering Number and Yield

Across all treatments, the tiller number followed the characteristic developmental pattern of increasing early in the season and declining thereafter (Figure 4). However, bixlozone-treated plants consistently produced fewer tillers than the other treatments at key growth stages, including the overwintering period, regreening phase, stem elongation, and heading, resulting in a reduced number of spikes (Figure 5a). The analysis of variance indicated no significant differences in grains per spike or thousand-grain weight among treatments (Figure 5b,c). Consequently, the bixlozone group exhibited the lowest final yield (Figure 5d). These findings illustrate that bixlozone notably suppresses the tiller formation and yield potential. This suppression likely stems from the herbicide’s inhibition of DXS, which impairs pigment biosynthesis, disrupts photosystem function, and reduces carbon assimilation [21]. These physiological disturbances limit the energy supply for tiller bud initiation and growth [22], ultimately causing the death of developing tillers.

The mixed treatment group exhibited improved tiller production, particularly during regreen stage II, and a significantly enhanced final yield compared with bixlozone alone (Figure 4 and Figure 5). This suggests that pyroxsulam partially offsets bixlozone-induced growth inhibition. One possible explanation is that pyroxsulam activates detoxification systems, such as glutathione S-transferases (GSTs) and ABC transporters [23], thereby accelerating bixlozone metabolism and reducing its effective toxicity.

2.2.3. Effects on Chlorophyll and Carotenoid Contents

In the treatment group at the field recommended dose (FRD), compared to the untreated control (CK), the single application of pyroxsulam (P) had no significant effect on the contents of the two pigments. However, the single application of bixlozone (B) caused a sharp decline in both the total chlorophyll and carotenoid contents of wheat leaves. By day 1 after treatment, the total chlorophyll content in group B was significantly reduced compared to CK. Pronounced phytotoxic effects were visible on days 2 and 4, with the chlorophyll contents in the B treatments dropping to approximately 22–30% and 20–25%, respectively, of those seen in CK, while the carotenoid contents exhibited a similar sharp decline. By day 7, the content of chlorophyll pigment in the bixlozone-only-treated groups remained at extremely low levels, with a slow recovery (Figure 6a,c). These symptoms are consistent with the established mode of action of bixlozone as an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a key enzyme involved in both the carotenoid and chlorophyll biosynthesis pathways [24]. Disruption of isopentenyl pyrophosphate (IPP) production results in reduced availability of precursors, resulting in carotenoid depletion, photodamage to chloroplast structures [25], chlorosis, and, ultimately, compromised photosynthetic activity [26,27].

In contrast, the mixed treatments (MIX) showed significantly higher chlorophyll and carotenoid contents across all measured time points compared to the single bixlozone treatments. Notably, by day 7, the chlorophyll content in the MIX group had returned to a level indistinguishable from CK, with significantly higher chlorophyll and carotenoid contents maintained. These results indicate that the mixed application preserved the necessary material basis and photo-protection barriers for photosynthesis, and effectively alleviated the degradation of photosynthetic pigments induced by bixlozone. The treatment group at 75% FRD showed the same trend (Figure 6b,d).

2.2.4. Chlorophyll Fluorescence Parameters Reveal Damage to PSII Function and Protective Effects

The treatment group of FRD showed that on days 1, 2, 4, and 7 after treatment, the maximum photochemical efficiency of PSII (Fv/Fm) in CK was stable at 0.78–0.84, indicating normal PSII function. The single treatment with pyroxsulam (P) had no significant effect on the Fv/Fm values. However, the single bixlozone treatment (B) caused a sharp decline in Fv/Fm. By day 2, the Fv/Fm value in group B dropped significantly to 0.347–0.539. The maximum damage was observed by day 4, with Fv/Fm values for Group B treatments all below 0.08, indicating near-complete loss of photochemical activity in the PSII reaction center. By day 7, the Fv/Fm values for the single bixlozone-treated groups were observed to have remained at extremely low levels. In contrast, the mixed treatments (MIX) showed a minimal effect on the PSII function. Their Fv/Fm values were consistently significantly higher than those of the corresponding single bixlozone treatments. For example, on day 2, the Fv/Fm value for MIX-treated plants (0.603–0.822) was markedly greater than that for Group B-treated plants. By day 7, the Fv/Fm values for mixed treatments had recovered to near-normal levels (Figure 7a).

The treatment group of 75% FRD showed the same trend with the FRD group (Figure 7b). At both concentrations, the addition of pyroxsulam significantly mitigated phytotoxicity and improved the wheat tolerance, with an entirely consistent trend and effect pattern. This indicates that the safening effect of pyroxsulam against bixlozone stress is stable and reproducible, rather than a random phenomenon observed at a single fixed dosage.

The experimental data indicate that the damage caused by bixlozone to the wheat photosynthetic system was not merely the destruction of a single link, but instead a progressive functional collapse along the linear electron-transport chain of photosynthesis. This process begins at the PSII reaction center. Initially, the sharp decline in chlorophyll and carotenoid contents weakens both the ability of the leaf to capture light energy and its potential for photo-protection, rendering the photosynthetic system more susceptible to damage by photo inhibitors. Starting from 48 h after treatment, a significant decrease in the maximum photochemical efficiency (Fv/Fm) of PSII indicates severe photoinhibition or photo-oxidative damage to the PSII reaction center.

Based on the analysis of the fluorescence parameters, we speculate that pyroxsulam effectively antagonized the initial photo-oxidative stress induced by bixlozone, thereby protecting PSII at the source and maintaining the normal operation of the entire photosynthetic chain from light energy conversion to carbon fixation.

2.3. Mechanisms Underlying Pyroxsulam-Mediated Enhancement of Wheat Tolerance to Bixlozone Stress at the Transcriptomic Level

RNA-seq was performed on 36 samples, producing 359.72 Gb of high-quality clean reads. Q30 scores exceeded 93.22% across all samples. After filtering, alignment to the Triticum aestivum reference genome (Available at: https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_018294505.1/ (accessed on 18 February 2026)) achieved mapping rates between 86.37% and 93.14%. Summary statistics for all samples are provided in Table S1.

2.3.1. Differentially Expressed Gene (DEG) Identification

To evaluate how herbicide combinations influence gene regulation, particularly the potential activation of detoxification pathways by pyroxsulam, transcriptomic data were analyzed to identify DEGs by using the widely adopted criteria of FDR < 0.05 and |log2FC| ≥ 2 [28,29,30]. A total of 65,000 annotated genes in the wheat genome were used as the background for calculating DEG proportions. Comparisons across the four treatment groups (CK: control, B: bixlozone alone, P: pyroxsulam alone, MIX: bixlozone + pyroxsulam) at 1, 2, and 4 days after treatment (DAT) revealed distinct temporal patterns in DEG numbers and proportions, with clear biological implications for the herbicide stress response and detoxification.

On day 1, intense transcriptional reprogramming was observed in all herbicide-treated groups compared to CK1—specifically, CK1_vs_B1 identified 10,114 DEGs (5970 upregulated and 4144 downregulated, accounting for 15.6% of total genes), and CK1_vs_MIX1 exhibited 6687 DEGs (3906 upregulated, 2781 downregulated, 10.3% of total genes) (Figure 8 and Figure 9a,b)—reflecting the immediate reaction of wheat to herbicide-induced stress, where the high DEG proportion in B1 indicates a more intense initial stress response to bixlozone alone, while the significantly lower number of DEGs in MIX1 than single treatments suggests synergistic antagonism that reduces redundant transcriptional reprogramming. This rapid gene activation, including the 3906 upregulated genes in CK1_vs_MIX1 enriched in xenobiotic degradation and glutathione metabolism, likely represents early detoxification or stress defense responses [31,32].

At DAT 2, DEG numbers and proportions declined across all groups—CK2_vs_B2 had 3104 DEGs (1709 upregulated, 1395 downregulated, 4.8% of total genes), and CK2_vs_MIX2 exhibited fewer DEGs (2664 DEGs, 1553 upregulated, 1111 downregulated, 4.1% of total genes) (Figure 9d,e)—and although the mixed-treatment MIX2 displayed fewer total DEGs, key regulatory genes (e.g., those involved in hormone signaling and metabolic adjustment) remained significantly altered, further supporting potential synergistic or antagonistic interactions between bixlozone and pyroxsulam that stabilize stress responses.

At day 4, CK4_vs_MIX4 had 2918 DEGs (1686 upregulated, 1232 downregulated, 4.5% of total genes), while CK4_vs_B4 had 5613 DEGs (4118 upregulated, 1495 downregulated, 8.6% of total genes), which was likely due to delayed accumulation of bixlozone metabolites triggering secondary stress (Figure 9g,h). The overall trend indicates progressive acclimation to herbicide stress [33], primarily including early stress-responsive genes, suggesting that detoxification and regulatory pathways may have stabilized or been compensated by other mechanisms [34]. The MIX group consistently maintained the lowest DEG proportion across all time points, confirming the herbicide combination’s role in mitigating prolonged cellular disturbance.

2.3.2. GO Enrichment Analysis

GO enrichment patterns showed that bixlozone alone strongly suppressed genes involved in photosynthesis, including those associated with carbon fixation (GO:0015977) and thylakoid membrane organization (GO:0010027), with clear quantitative support from enrichment analysis (Figure 10): in the CK_vs_B comparison, photosystem II oxygen evolving complex (GO:0009654)—a core component of photosynthetic electron transport—exhibited an enrichment factor of 4.39 and a corrected p-value (q value) of 4 × 10⁻⁶ (extremely significant), involving 17 genes with mixed up/downregulation (predominantly downregulated, consistent with suppressed photosynthetic function); thylakoid membrane organization (GO:0010027) had an enrichment factor of 3.39 (q value = 0.190) and 4 associated genes; chlorophyll binding (GO:0016168) showed an enrichment factor of 1.75 (q value = 0.159) with 11 genes; and carbon fixation (GO:0015977) had an enrichment factor of 0.39 (q value = 0.878). These changes are consistent with the visible chlorosis observed phenotypically and indicate that wheat under bixlozone stress diverts gene expression toward defense, rather than detoxification.

In contrast, pyroxsulam alone enhanced the expression of genes linked to detoxification functions, with two key Gene Ontology (GO) terms explicitly enriched: GO:0016705 (oxidoreductase activity, acting on paired donors, with incorporation or reduction in molecular oxygen), which was associated with xenobiotic degradation and ROS scavenging, showed significant enrichment in the CK4_vs_P4 comparison (enrichment factor = 1.55, q value = 0.0026, 76 associated genes). By comparison, the same term was non-significant in bixlozone alone (CK4_vs_B4: enrichment factor = 0.94, q value = 0.714, 78 genes) and less significant in the mixed treatment (CK4_vs_MIX4: enrichment factor = 1.43, q value = 0.029, 63 genes); GO:0005215 (transporter activity), which was related to herbicide sequestration and metabolite transport, was weakly enriched in CK4_vs_P4 (enrichment factor = 0.75, q value = 0.756, 2 genes) but showed no significant enrichment in other groups (e.g., CK4_vs_B4: enrichment factor = 1.55, q value = 0.320, 7 genes).

This transcriptional activation of GO:0016705 with the lowest q value (0.0026) in pyroxsulam alone agrees with previous findings indicating that detoxification-related pathways can be induced in response to herbicide stress [35]. Notably, the mixed treatment produced a shift in enrichment patterns: instead of the photosynthesis-related suppression observed in the bixlozone group, the mixture resembled the detoxification-associated signature triggered by pyroxsulam alone (e.g., GO:0016705 q value = 0.029 in MIX vs. 0.0026 in P). This suggests that pyroxsulam may alleviate bixlozone toxicity by stimulating metabolic pathways that are capable of degrading or sequestering bixlozone or its harmful intermediates. This is consistent with previous findings suggesting that exogenous substances can induce detoxification and metabolic processes in plants against herbicides [36].

2.3.3. KEGG Enrichment Analysis

KEGG pathway enrichment provided additional mechanistic insight that was complementary to the GO analysis. In the bixlozone-only treatment, DEGs were predominantly enriched in pathways related to photosynthesis, porphyrin and chlorophyll metabolism, and carotenoid biosynthesis (Figure 11). The results of pathway enrichment analysis showed that after the mixed treatment of pyroxsulam and bixlozone, the differentially expressed genes in wheat were significantly enriched in pathways related to energy synthesis and metabolism, suggesting that their mixed application may exert potential effects on cellular energy synthesis and metabolic processes in wheat. For plants treated solely with pyroxsulam, significantly enriched pathways included the plant–pathogen interaction and biodegradation of drugs pathways, suggesting that wheat strongly activated its detoxification machinery in response to pyroxsulam exposure, in agreement with its role as a broad-spectrum herbicide [37]. In the mixed treatment, genes involved in the glutathione (GSH) metabolic pathway (ko00480) and ABC transporter systems (ko02010) were significantly upregulated. These pathways play central roles in xenobiotic metabolism: the former constitutes the core intracellular biochemical process for antioxidant defense and detoxification, while the latter refers to the active transmembrane transport of toxic substances driven by ATP consumption. It is speculated that pyroxsulam may attenuate bixlozone toxicity by inducing glutathione-conjugation reactions and active transport processes that facilitate the efflux or compartmentalization of bixlozone or its metabolites.

It should be noted that the lack of time-series statistical modeling is a limitation of the current experimental design. In subsequent studies, we will optimize the design by adding a continuous time gradient (e.g., DAT1–DAT7 with daily intervals) and adopt STEM time-series analysis to construct a time × treatment interaction model, thereby exploring the dynamic trends of DEGs and the temporal molecular mechanisms underlying pyroxsulam-mediated phytotoxicity alleviation.

2.3.4. qRT-PCR Validation

The expression levels of seven candidate genes identified by RNA sequencing were validated using qRT-PCR. The results showed that all seven candidate genes were significantly regulated, which was consistent with the RNA sequencing data. Pyroxsulam treatment significantly upregulated the expression of TraesCS3B03G0337400 (ABCC8-X1) (Figure 12a), TraesCS1A03G0501900 (GSTU6) (Figure 12b), and TraesCS7D03G0260400 (ABCC10) (Figure 12c). ABCC8-X1 and ABCC10 belong to the ABCC subfamily of ATP-binding cassette (ABC) transporters, which are well-documented as playing core roles in plant detoxification by mediating the transport and sequestration of xenobiotics (e.g., herbicides) and their conjugated metabolites (e.g., glutathione-conjugates) [38]. Homologs of ABCC8 in plants have been directly linked to herbicide resistance: for instance, overexpression of ABCC8 in Echinochloa colona confers glyphosate resistance by extruding the herbicide from cells or sequestering it into vacuoles to reduce intracellular toxicity [39]. Similarly, ABCC10 is characterized as a broad-spectrum xenobiotic transporter in eukaryotes that is capable of exporting diverse toxic substrates (including drug molecules and hormone conjugates) via ATP-dependent transport. In plants, ABCC10 homologs are induced under herbicide stress and cooperate with glutathione S-transferases (GSTs) to enhance detoxification—GSTs first conjugate herbicides with glutathione to form hydrophilic complexes, which are then specifically recognized and transported by ABCC transporters for extracellular excretion or vacuolar sequestration [13]. This result confirms the previous findings that detoxification-related genes play an important role in plant responses to environmental stressors [13,40,41].

Notably, two ABC transporters in Chlamydomonas reinhardtii have been reported to participate in molybdenum (Mo) transport [42], though studies on the correlation between wheat ABC transporters and Mo transport remain scarce. We speculate that Mo may indirectly contribute to wheat’s detoxification of bixlozone: as an essential micronutrient, Mo is a core component of the Mo-containing enzymes (e.g., nitrate reductase) involved in stress responses and metabolic detoxification, and the ABC transporters identified herein (e.g., ABCC8-X1, ABCC10) might regulate Mo transport to target tissues/organelles, thereby affecting Mo enzyme activity and detoxification metabolism. However, Mo content, distribution, and Mo enzyme activity were not measured in this study, which will be supplemented in future research to clarify these associations.

Additionally, nitric oxide (NO) acts as a key second messenger in plants, and nitrate reductase (a critical Mo-containing enzyme) is a major pathway for plant NO synthesis. We hypothesize that NO may mediate the alleviation of bixlozone phytotoxicity by pyroxsulam: bixlozone stress may inhibit nitrate reductase activity and reduce NO production, while pyroxsulam could mitigate this inhibition, and NO may further regulate the expression of ABC transporters and detoxifying enzymes (e.g., GSTs) via downstream signaling pathways (e.g., MAPK) or balance reactive oxygen species (ROS) to reduce oxidative damage. Subsequent studies will detect NO content and nitrate reductase activity to verify this hypothesis.

Treatment with bixlozone also markedly induced the upregulation of these three genes, suggesting their potentially significant role in the detoxification metabolism of bixlozone in wheat. Mixed treatment with pyroxsulam and bixlozone induced the expression of these three genes to an even higher level. This observation is consistent with the changes in phytotoxicity symptoms: wheat possesses a certain inherent capacity to detoxify bixlozone, but this capacity is insufficient to completely eliminate its effects, resulting in observable phytotoxicity symptoms. However, when pyroxsulam is added, the detoxification metabolism of bixlozone in wheat is enhanced, leading to a significant alleviation of phytotoxicity symptoms.

Conversely, genes such as TraesCS3D03G0791000 (MRP2) (Figure 12d) were significantly downregulated in the mixed treatment, potentially indicating a redistribution of metabolic resources away from certain transport processes under mixed herbicide exposure. This type of regulatory rebalancing has frequently been described in studies examining the growth–defense trade-off under stress. Other genes, including TraesCS5B03G1170400 (ABCC8) (Figure 12e), TraesCS6D03G0359400 (MRP) (Figure 12f) and TraesCS5D03G0584600 (GST23) (Figure 12g), displayed variable expression patterns across treatments. Such fluctuating dynamics suggest that multiple transporters participate in a coordinated regulatory network during mixed-herbicide exposure: a pattern that is consistent with prior reports on complex stress-induced transcriptional responses [43].

2.4. Implications of the Results for Further Research Work

Bixlozone is a proherbicide that requires in plant activation [44]. Its efficacy may vary due to impaired conversion to 5-ketobixlozone and/or enhanced metabolism into low-toxicity metabolites. Studies have shown [45] that wheat tolerance to bixlozone correlates poorly with activation of the herbicide. Therefore, wheat tolerance to bixlozone depends primarily on detoxification metabolism. Based on the integration of the present transcriptomic data and qRT-PCR validation, we suggest that pyroxsulam enhances its wheat tolerance to bixlozone by inducing a detoxification system dominated by GSTs and ABC transporters—where GSTs mediate glutathione conjugation of bixlozone or its toxic metabolites to form hydrophilic complexes, and ABC transporters (e.g., ABCC8-X1, ABCC10) potentially facilitate the efflux of these conjugates out of cells or their vacuolar sequestration. Activation of these pathways establishes a biochemical “buffer” that reduces cellular exposure to bixlozone. Under mixed treatment, this pyroxsulam-induced detoxification network appears capable of processing not only pyroxsulam itself but also detoxifying bixlozone or its reactive intermediates. This indicates that pyroxsulam stimulates a broad-spectrum detoxification response, rather than acting through simple antagonistic interactions. The resulting system enables wheat to metabolize both ALS-inhibiting herbicides and DXS-inhibiting herbicides concurrently, thereby reducing phytotoxic damage. These findings support the broader principle that the activation of endogenous detoxification systems can mitigate herbicide injury in mixture applications and additionally reveal a distinctive protective role of pyroxsulam. Rather than relying on post-damage repair, pyroxsulam primes the plant for enhanced xenobiotic metabolism. This mechanistic insight provides a valuable foundation for targeted gene-editing strategies aimed at improving crop stress resilience.

However, it is important to acknowledge several key limitations of the present study: while transcriptional induction of these detoxification-related genes (GSTs, ABC transporters) is consistently observed, direct functional validation of their roles in bixlozone metabolism or sequestration is lacking. To address this gap and deepen the mechanistic understanding, future studies will focus on rigorous gene function verification, including heterologous expression assays to confirm the catalytic activity of candidate GSTs in bixlozone conjugation, and gene silencing (e.g., RNAi) or overexpression experiments to validate the role of ABC transporters in bixlozone efflux or vacuolar sequestration. These follow-up investigations will help to solidify the causal relationship between gene expression changes and the observed safening effect, further refining the molecular mechanism of pyroxsulam-mediated phytotoxicity alleviation.

In addition, only one wheat cultivar, Jimai 22, was used in this study. Herbicide tolerance and safener responsiveness are known to vary among wheat genotypes, and different cultivars may exhibit distinct detoxification capacities and regulatory mechanisms. Therefore, the conclusions obtained in this study are preliminary and may be cultivar-dependent. Further studies involving a wider range of wheat germplasms with diverse genetic backgrounds are needed to verify the universality of the safening effect of pyroxsulam on bixlozone phytotoxicity. In addition, the effectiveness of this herbicide–safener combination under different environmental conditions, such as variable temperature, soil moisture, and fertility, also warrants further investigation to ensure its stability and reliability in practical field production.

Thirdly, under controlled indoor conditions, both 100% FRD and 75% FRD of bixlozone caused significant phytotoxicity and growth inhibition in wheat. At both concentrations, the addition of pyroxsulam significantly mitigated phytotoxicity and improved wheat tolerance, with an entirely consistent trend and effect pattern. This indicates that the safening effect of pyroxsulam against bixlozone stress is stable and reproducible, rather than a random phenomenon observed at a single fixed dosage. It provide sufficient support for the mechanistic discussion, ensuring the reliability of the conclusions and their practical guidance for field production. A comprehensive dose–response curve and detailed comparative analysis among different application rates will be conducted in further research.

3. Materials and Methods

3.1. Herbicide Control Efficacy to Weeds

To address whether the pyroxsulam addition affects the control efficacy of bixlozone against A. myosuroides, field experiments were carried out in Nanhe District, Xingtai City, Hebei Province (40°26′46″ N, 79°58′56″ W). The soil at the site is loam with moderate organic matter content. Wheat was sown on 10 October 2023, following rotary tillage, at a rate of 225 kg/ha. The wheat variety was Jimai 22, a high-yielding, stable-yielding and widely adaptable winter wheat cultivar that is widely distributed and cultivated on a large scale in China. A randomized block design was implemented, with four replicates per treatment and plot areas of 40 m². Herbicide applications were performed on 17 November 2023. The control efficacy was assessed on 7 March 2024. Four sampling points (0.25 m² each) were selected per plot, and the number of tillers and the aboveground fresh weight of all A. myosuroides plants within each sampling point were recorded. To ensure that the findings provide practical guidance for agricultural production and comply with the general protocols for field efficacy trials, we applied the commercial herbicide formulations in the field with the following treatments:

CK-F (control): No herbicide applied; weeds removed manually.

P-F (pyroxsulam alone): A total of 4% pyroxsulam oil dispersion (Corteva Agriscience LLC, Indianapolis, IN, USA), applied at 15 g a.i./ha.

B-F (bixlozone alone): A total of 36% bixlozone suspension concentrate (FMC Corporation, Philadelphia, PA, USA), applied at 216 g a.i./ha.

B-F_l (low dosage bixlozone alone): A total of 36% bixlozone suspension concentrate (FMC Corporation, USA), applied at 162 g a.i./ha.

MIX-F (mixed application): Bixlozone (216 g a.i./ha) + pyroxsulam (15 g a.i./ha), applied simultaneously.

MIX-F_l (low dosage bixlozone mixed application): Bixlozone (162 g a.i./ha) + pyroxsulam (15 g a.i./ha), applied simultaneously.

3.2. Effect of Pyroxsulam on Bixlozone Phytotoxicity in Field Conditions

The experimental design was consistent with that described in Section 3.1. As the phytotoxicity of bixlozone at a dose of 216 g a.i./ha was relatively mild under field conditions, phytotoxicity assessments were conducted only for the following four treatments: CK-F, P-F, B-F, and MIX-F. For the morphological assessment, there were three sampling points per plot, with 20 plants per point to record the tiller number. Yield components, including the number of spikes per square meter, grains per spike, thousand-grain weight, and grain yield, were measured at maturity.

Tiller measurements were conducted on 7 December 2023 (pre-overwintering), and on 7 March 2024 (regreen stage I), 15 March 2024 (regreen stage II), 29 March 2024 (jointing stage), and 18 April 2024 (booting stage). The final yield was recorded at harvest on 10 June 2024.

To assess herbicide-induced phytotoxicity, we referred to the current national and industrial standards for phytotoxicity classification in China, specifically the relevant provisions of the Pesticide—Guidelines for the Field Efficacy Trials (I)—Herbicides against Weeds in Cereals (GB/T 17980.41-2000) [46] guideline and the Guideline for Laboratory Bioassay of Pesticides—Part 8: Crop Safety Test (NY/T 1155.8-2007) [47]. Combined with the actual phytotoxic symptoms of wheat caused by bixlozone observed in this experiment (e.g., leaf chlorosis, wilting, severity of growth inhibition, etc.), the criteria underlying injury grades are described below:

Grade 0: No visible damage; normal plant growth.

Grade 1: Herbicide spot area representing 1–10% of the plant or region.

Grade 2: Herbicide spot area representing 11–30% of the plant or region.

Grade 3: Herbicide spot area representing 31–50% of the plant or region.

Grade 4: Herbicide spot area exceeding 51% of the plant or region.

Each plot was evaluated and graded on 10 December 2023.

3.3. Symptoms of Herbicide Damage During Indoor Cultivation

Before initiating the laboratory trial, potting soil was added to each container to approximately 2 cm below the rim and irrigated thoroughly using a fine-mist nozzle until water drained freely from the bottom. After excess moisture had drained, uniformly sized, fully mature wheat seeds of the Jimai 22 were distributed on the soil surface and covered with a 1 cm layer of soil. Pots were then transferred to a controlled-environment growth chamber set at 20 °C with a 12 h light/12 h dark cycle.

Seedlings were thinned and subjected to herbicide treatments once they reached the one-leaf stage, approximately 7 days after sowing. Each pot was adjusted to retain 15 vigorous, uniformly growing seedlings, while any irregularly sized or malformed individuals were removed. Solutions were sprayed using a research track sprayer (3WP-2000, Nanjing Institute of Agricultural Mechanization, Ministry of Agriculture and Rural Affairs, Nanjing, China), delivering 450 L/ha spray solution at 0.3 MPa. To ensure that the results specifically reflect the biological effects of the active ingredients and improve the accuracy and reliability of mechanistic analysis, we used the technical grade herbicides in the laboratory with the following treatments:

P (pyroxsulam alone): A total of 98% pyroxsulam technical material (Shandong Austro-kun Crop Science Co., Ltd., Jinan, China), applied at 15 g a.i./ha.

B (bixlozone alone): A total of 96% bixlozone technical material (FMC Corporation, USA), applied at 216 g a.i./ha.

B_l (low dosage bixlozone alone): A total of 96% bixlozone technical material (FMC Corporation, USA), applied at 162 g a.i./ha.

MIX (mixed application): Bixlozone (216 g a.i./ha) + pyroxsulam (15 g a.i./ha), applied simultaneously.

MIX_l (low dosage bixlozone): Bixlozone (162 g a.i./ha) + pyroxsulam (15 g a.i./ha), applied simultaneously.

CK (untreated control): Received an equal volume of distilled water.

Each treatment was replicated three times, with 15 seedlings per replicate. Sampling occurred at 24 h (prior to visible injury), 48 h (symptom development phase), and 96 h (symptom stabilization) after herbicide exposure. Leaf coloration and morphology were regularly documented. Herbicide injury was assessed using the grading method described in Section 2.1.

3.4. Determination of Plant Carotenoid and Chlorophyll Contents

Wheat samples were treated as described in Section 3.3. The levels of carotenoids and chlorophyll in fresh leaves were measured at 24, 48, and 96 h after treatment, using a Plant Carotenoid Assay Kit (Beijing Solabao Technology Co., Ltd., Beijing, China) and a Plant Chlorophyll Content Assay Kit (Beijing Solabao Technology Co., Ltd., Beijing, China), respectively, with absorbances read using a microplate reader (Agilent BioTek Synergy H1, Santa Clara, CA, USA).

3.4.1. Sample Preparation

Fresh plant leaves were selected, washed thoroughly with distilled water, and dried to remove surface moisture, and areas such as the midrib which were not used for analysis were removed. Pre-weighed samples (0.1 g) were mixed separately with either 1.5 mL of chlorophyll or carotenoid extraction solution, followed by the addition of 10 mg of the respective reagent 1; the mixture was then thoroughly ground in dim light to avoid photodegradation. The homogenate was transferred to 2 mL EP tubes wrapped in tin foil, and incubated at room temperature for 30 min, during which the tubes were inverted gently three times to enable mixing and enhance the extraction efficiency. The mixture was then centrifuged at 4000 rpm for 5 min at room temperature, and the supernatant was retained for further analysis. Measurements were performed immediately after extraction to ensure data accuracy and prevent evaporation of the extract or chlorophyll degradation.

3.4.2. Determination of Carotenoid Contents

The microplate reader was preheated for 30 min, with sequential adjustments of the wavelengths to 470 nm, 646 nm, and 663 nm. The 200 µL aliquots of the supernatant were added to the wells of a 96-well plate, and the absorbance values at the three wavelengths (A_470-ca, A_646-ca, A_663-ca) were measured in succession.

3.4.3. Determination of Chlorophyll Content

The microplate reader was preheated for 30 min, with wavelengths set to 645 nm and 663 nm. The 200 µL aliquots of the supernatant were added to the wells of a 96-well plate, and absorbance values at the two wavelengths (A_645-ch and A_663-ch) were measured.

3.4.4. Calculation of Carotenoid Content

The following formulae were used to calculate the carotenoid concentrations:

C_a(mg/L) = 12.21 × A_663-ca − 2.81 × A_646-ca

C_b(mg/L) = 20.13 × A_646-ca − 5.03 × A_663-ca

Carotenoid concentration:

C_c(mg/L) = (1000 × A_470-ca − 3.27 × C_a − 104 × C_b) ÷ 229

Carotenoid content (mg/g):

Carotenoid content (mg/g) = C_c × V _extraction × F_ca ÷ W_ca

where

• A_470-ca, A_646-ca, and A_663-ca are the absorbances at the respective wavelengths.
• F_ca is the dilution factor.
• W_ca is the sample mass (g).

3.4.5. Calculation of Chlorophyll Content

The formulae for determining the chlorophyll contents were as follows:

Chlorophyll a content (mg/g):

Content of chlorophyll a (mg/g) = 0.0015 × (21.2 × A_663-ch − 4.48 × A_645-ch) × F_ch ÷ W_ch

Chlorophyll b content (mg/g):

Content of chlorophyll b (mg/g) = 0.0015 × (38.2 × A_645-ch − 7.8 × A_663-ch) × F_ch ÷ W_ch

Total chlorophyll content (mg/g):

Content of total chlorophyll (mg/g) = 0.0015 × (33.7 × A_645-ch + 13.4 × A_663-ch) × F_ch ÷ W_ch

where

• A_645-ch and A_663-ch are the absorbances at the respective wavelengths.
• F_ch is the dilution factor.
• W_ch is the sample mass (g).

3.5. Measurement of Photosynthetic Traits

At 24, 48, and 96 h after treatment, the minimal fluorescence (F0) and maximal fluorescence (Fm) were measured at the chlorophyll-chlorotic sites of wheat leaves in each treatment group using a PAM-2500 portable chlorophyll fluorescence analyzer (WALZ, Bavaria, Germany), and the maximum photochemical efficiency of PSII (Fv/Fm) was calculated. Three replicates were included in each treatment.

3.6. RNA-Seq and Identification of Candidate Genes

To investigate the transcriptional response of genes under different treatments, RNA-seq analysis was performed on wheat samples across three time points (1, 2, and 4 days after treatment), with four treatment groups (CK, P, B, MIX) and three biological replicates per group at each time point (as detailed in Section 2.2). RNA was isolated from plant tissues using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). RNA concentration, purity, and integrity were assessed using a NanoDrop 2000 spectrophotometer and an Agilent 2100 Bioanalyzer. High-quality RNA (≥1 µg) was used for library preparation with the Hieff NGS Ultima Dual-Mode mRNA Library Prep Kit (Yeasen Biotechnology, Shanghai, China). Adapter ligation, PCR amplification, and size selection were performed following the standard protocols. Sequencing was conducted on an Illumina NovaSeq platform to generate 150 bp paired-end reads. Clean reads were aligned to the reference genome (Triticum aestivum v2.1), using HISAT2.

Given the small number of time points (three discrete time points without a continuous gradient), the current experimental design is not optimal for complex time-series statistical models (e.g., STEM analysis, mixed linear models) that dissect time × treatment interaction effects. Thus, the differentially expressed gene (DEG) analysis performed herein is descriptive, rather than dynamic, time-series analysis, with its core purpose being to screen genes with significant differential expression between key comparison groups (CK vs. B, B vs. MIX) at each time point. DESeq2 (Version 1.30) software was used to compare the different treatments with control samples to identify differentially expressed genes (DEGs). Blast2GO was used to classify DEGs, using the criteria of p-value < 0.05 and absolute fold change ≥2 for DEG identification. The clusterProfiler package in R was used to perform gene enrichment analysis [48], and KEGG (http://www.genome.jp/kegg/) (accessed on 19 November 2025) was used to analyze the pathways associated with the DEGs.

To verify the RNA-seq data, seven detoxification-related differentially expressed genes (DEGs) were selected for expression analysis using quantitative real-time PCR (qRT-PCR), according to the following screening criteria: (1) the genes are closely associated with herbicide detoxification or stress tolerance; (2) the genes exhibit significantly differential expression between the pyroxsulam + bixlozone treatment and the bixlozone alone treatment (|log₂(fold change)| > 2, adjusted p-value < 0.05); and (3) the genes possess clear functional annotations in public databases related to the research focus. The TruScript 1st Strand cDNA synthesis kit (Aidlab Biotechnologies Co., Ltd., Beijing, China) was used to synthesize cDNA in a 20 μL reaction system. Specific primers (Table S2) were designed using Oligo 7 (Molecular Biology Insights, Colorado Springs, CO, USA) and a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR. The Triticum aestivum reference genome (v2.1) served as an internal reference, and the relative gene expression levels were calculated using the ^2−ΔΔCT method. All experiments were conducted with three biological and three technical replicates.

3.7. Statistical Analyses

Analysis of variance was used to determine the significance of treatment and time points, and t-tests in SPSS 25.0 (IBM Corp., Armonk, NY, USA) were used to determine significant differences between means, with p <0.05 considered statistically significant. GraphPad Prism 9 was used for graphics.

4. Conclusions

This study combined detailed phenotypic evaluation with transcriptomic profiling to elucidate the effect of pyroxsulam to enhance the tolerance of wheat to bixlozone and its associated mechanisms. In terms of phytotoxicity symptoms, the mixed application of pyroxsulam markedly lessens bixlozone-related chlorosis and reduces negative effects on tillering and yield. Regarding physiological indicators, pyroxsulam alleviated the bixlozone-induced dysfunction of the PSII reaction center (as evidenced by reduced Fv/Fm) and the degradation of chlorophyll and carotenoid. Transcriptomic analysis revealed that across all sampling times (1, 2, and 4 days), herbicide treatments produced significant and time-dependent shifts in transcriptional profiles. Under bixlozone stress, pyroxsulam-induced detoxification pathways played a dominant protective role, establishing a direct molecular basis for the observed mitigation of injury. qRT-PCR assays further confirmed that pyroxsulam initiates a signaling cascade that induces detoxification genes such as ABCC8-X1, GSTU6 and ABCC10. Collectively, the findings indicate that rather than acting through physical or chemical antagonism, pyroxsulam functions as a molecular inducer, triggering the expression of detoxification-associated genes that accelerate bixlozone metabolism and sequestration, and thereby reducing its phytotoxic impact. This work advances the understanding of herbicide interactions and provides a strong theoretical platform for optimizing pyroxsulam–bixlozone combinations. It also offers valuable insights and genetic targets for breeding or engineering crops with enhanced herbicide tolerance. Ultimately, the study contributes foundational knowledge for developing safer, more efficient herbicide management strategies within sustainable agricultural systems.