BX6-dependent benzoxazinoid biosynthesis enhances herbivore resistance and salt stress tolerance in durum wheat Triticum turgidum

BX6-dependent benzoxazinoid biosynthesis enhances herbivore resistance and salt stress tolerance in durum wheat Triticum turgidum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article BX6-dependent benzoxazinoid biosynthesis enhances herbivore resistance and salt stress tolerance in durum wheat Triticum turgidum Let Kho Hao, Reut Shavit, Beery Yaakov, Adi Kliot, Murad Ghanim, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9235518/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Benzoxazinoids (BXDs) are a class of indole-derived specialized metabolites with primarily defensive roles against herbivores and are found in both monocots and eudicots. We recently demonstrated that BXDs contribute to drought tolerance and aphid resistance in hexaploid wheat. In this study, we investigated the potential roles of BXDs in tetraploid wheat resistance to herbivores with different feeding modes and in tolerance to salt stress using a stable BXD-deficient mutant. We first observed accumulation of BXDs upon insect herbivory and salt stress in wheat. To investigate BXD's function, we knocked out the key biosynthetic gene, BX6 , in tetraploid wheat ( Triticum turgidum cv. Svevo) using CRISPR-Cas9. BX6 deficiency affected herbivore performance depending on feeding mode: sucking herbivores like aphids and two-spotted spider mites performed better on mutant plants than on wild-type plants, while chewing herbivores like moth caterpillars' growth was unaffected. Under salt stress, the mutant plants showed a significant reduction in total chlorophyll, biomass, and water content in both leaves and roots compared to wild-type plants. Additionally, salt-stressed mutant plants had higher levels of electrolyte leakage and hydrogen peroxide compared to the wild type, indicating aggravated cell membrane damage and elevated oxidative stress, likely due to impaired detoxification of reactive oxygen species. These findings suggest that BX6-derived BXDs are essential for wheat herbivore resistance and salt stress tolerance. This study expands our understanding of the multifaceted roles of BXDs in stress resilience and highlights their potential for improving plant adaptation to environmental challenges and climate change. Plant defense stress resilience benzoxazinoids BX6 herbivore resistance Triticum turgidum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message Functional loss of BX6 reveals benzoxazinoids as crucial mediators of wheat herbivore resistance and salt tolerance, linking specialized metabolism to environmental resilience. Background Wheat ( Triticum spp.) is an economically and nutritionally important cereal crop, which occupies around 17% of the global cultivated area. Moreover, it provides food for about 35% of the world's population and approximately 20% of humanity's consumed calories [ 1 , 2 ]. Wheat production is adversely affected by both environmental stress and biotic stress. Salinity is one of the most severe environmental stresses, significantly reducing crop yield and quality worldwide. It is estimated that over 20% of cultivated land—responsible for producing one-third of the world's food—is affected by soil salinity, reducing productivity [ 3 , 4 ]. Salt stress has a strong effect on plant growth and development. Salinity disrupts cellular ultrastructure, impairs the photosynthetic apparatus, damages membranes, elevates reactive oxygen species (ROS) production, and reduces enzymatic activity, collectively limiting crop growth and yield [ 5 , 6 ]. Biotic stress due to herbivore pest infestation significantly reduces food quality and crop yield [ 7 ]. Aphids (order Hemiptera, family Aphididae) are major pests of wheat and other grass family species [ 8 ]. Aphids consume water and nutrients from the phloem sap of plants, which poses both osmotic and chemical challenges due to its high solute concentration and defensive metabolites [ 9 ]. Aphid feeding on plants causes substantial yield losses through direct damage and indirectly, through facilitation of secondary infections. Furthermore, aphids transmit nearly 40% of all known plant viruses, including several of the most destructive viral diseases in wheat, such as the wheat yellow mosaic virus and wheat streak mosaic virus [ 10 , 11 ]. Other notable pests of wheat include leaf-chewing insects such as caterpillars of the Lepidoptera order. Caterpillar infestation triggers extensive transcriptomic reprogramming in plants and production of specialized defensive metabolites [ 12 – 14 ]. While plant responses to aphids and caterpillars in grass crops are relatively well studied, the impact of mite herbivory is less so, despite them being prominent wheat pests, especially in warm climates. However, recent studies have shown that the two-spotted spider mite (TSSM; Tetranychus urticae ) can significantly damage cereal crops, including maize, rice, barley, and wheat, highlighting its emerging importance as a pest [ 15 , 16 ]. Abiotic and biotic stresses, which negatively impact crop production, are expected to increase due to global climate change [ 17 , 18 ]. To overcome challenges imposed by these stressors, plants have evolved arrays of mechanisms, including specialized metabolite production. Plant specialized metabolites (PSMs), also known as secondary metabolites, are diverse chemical compounds that play vital roles in plant responses to various stresses. PSMs were shown to function more in plant defense rather than in essential processes like growth and reproduction [ 19 ], with over 200,000 known PSMs produced by plants [ 20 ]. Perhaps the most known function of PSMs is acting as antifeedants against arthropod herbivores; targeting the pests' nervous, digestive, and endocrine systems [ 21 ]. PSMs also play several key roles in abiotic stress tolerance, such as antioxidant defense, membrane stabilization, metal chelation, and UV protection [ 22 ]. Benzoxazinoids (BXDs) are indole-derived specialized PSMs abundantly produced in Poaceae crops such as maize ( Zea mays ), wheat ( Triticum spp.), and rye ( Secale cereale ) [ 23 ]. BXDs' biosynthesis begins in the plastids with indole-3-glycerol phosphate lyase benzoxazinoneless 1 (BX1), converting indole-3-glycerol phosphate into indole, followed by cytochrome P450 enzymes (BX2-5), forming the core compound 2,4-dhydroxy-1,4-benzoxazin-3-one (DIBOA). DIBOA is stabilized through glycosylation by BX8 and BX9 and stored as DIBOA-Glc in the vacuole to prevent autotoxicity [ 24 ]. Further modifications, by tailoring enzymes such as BX6 (a dioxygenase) and BX7 (an O -methyltransferase), convert DIBOA-Glc into DIMBOA-Glc, the most abundant form of BXDs in wheat and maize [ 25 ]. Additional derivatives such as HDMBOA-Glc, TRIMBOA-Glc, DIM 2 BOA-Glc, and HDM 2 BOA-Glc are produced through sequential tailoring enzymatic steps by additional O -methyltransferases (BX10-12 & 14) and dioxygenase (BX13). To date, genes encoding BX6 and BX10 are the only tailoring enzyme genes that have been identified and characterized in wheat [ 26 – 28 ], whereas those corresponding to BX7, BX11, BX12, BX13, and BX14 have not yet been discovered. Owing to their abundance and essential roles in economically and nutritionally important crops, BXDs have been extensively studied for over 50 years [ 29 ]. BXDs are primarily known for defense against various biotic stresses, including insect herbivores, microbial pathogens, and competing plant species [ 12 , 30 , 31 ]. BXDs have been reported to affect the fitness of phloem feeding insects such as aphids [ 32 – 35 ], chewing herbivores such as caterpillars [ 14 , 36 , 37 ], cell content feeders such as spider mites [ 38 ], and pathogens such as fungi [ 13 ]. Shavit et al. [ 27 ] reported that silencing the wheat dioxygenase, TaBX6 , using virus-induced gene silencing (VIGS) increased wheat susceptibility to both aphids and spider mites, while caterpillar performance was unaffected. This indicates that TaBX6 plays a role in plant defense against sucking herbivores. Besides acting as direct defense compounds (toxins), BXDs, particularly DIMBOA-Glc or its aglucon DIMBOA, also function as regulators for other defensive processes, such as callose deposition [ 26 , 35 , 39 ]. Changes in the levels of BXDs upon environmental stresses have also been reported. For example, drought stress increases the levels of DIMBOA and DIBOA in maize [ 40 ], and the O -methyltransferase ZmBX12, which converts DIMBOA-Glc into HDMBOA-Glc, has been linked to drought adaptation in this plant [ 41 ]. In addition, Poschenrieder et al. [ 42 ] demonstrated that adding DIMBOA to the growth medium of an aluminum-susceptible maize line is sufficient to protect the plants from the adverse effects of aluminum, suggesting its potential role in safeguarding maize plants against aluminum toxicity. In wheat, Batyrshina et al. [ 43 ] reported that environmental stresses such as drought, salinity, and cold affected the levels of BXDs in the leaves of wheat seedlings. These findings highlight a potential, unexplored role for BXDs in abiotic stress tolerance. Recently, we demonstrated that drought triggers BXD accumulation and callose deposition in the leaves, which disrupt aphid feeding, supporting a dual role for BXDs in hexaploid wheat ( Triticum aestivum ) drought tolerance and aphid resistance [ 44 ]. Here, we investigate how wheat modulates BXD production in response to herbivory or salt stress and examined their roles in conferring herbivory and salt tolerance resistance using tetraploid wheat ( Triticum turgidum ). Understanding how biotic and abiotic stresses alter BXD profiles and how these changes contribute to plants' stress tolerance is crucial for developing sustainable approaches to address upcoming agricultural challenges. Materials and methods Plant material, insect colony, and growth room conditions Seeds of Triticum turgidum ssp. durum cultivar Svevo (SV) or BXD-deficient mutants were germinated on moistened Whatman filter paper. SV wheat seeds were obtained from laboratory of Prof. Assaf Distelfeld (Haifa University, Isarel), while BXD-deficient mutants were generated in the laboratory of Prof. Vered Tzin (Ben-Gurion University of Negev, Israel). Two days after sowing, transplanted into individual pots containing a tuff-vermiculite mixture (2:1) supplemented with N-P-K fertilizer (20–20–20). Plants were grown in a controlled growth room under 16 h light/8 h dark cycle; 250–350 µmol photons m − 2 s − 1 light intensity from a 3000-lm LED, 22 ± 3°C; and 60 ± 10% relative humidity. Soil moisture was maintained by watering all pots equally for two days after transplanting [ 27 , 43 – 45 ]. As BXDs are abundant in young wheat leaves but decline during plant development [ 44 – 46 ], all experiments were performed on 14-day-old seedlings under the same growth room conditions unless otherwise stated. A colony of bird cherry-oat aphids ( Rhopalosiphum padi ) was maintained on bread wheat seedlings ( T. aestivum cv. Rotem) as previously described [ 44 , 45 ]. For all aphid bioassays, approximately two-week-old apterous adults were used. A colony of Two- Spotted Spider Mites (TSSM, Tetranychus urticae ) were reared on Capsicum annum plants (commercial line 'Ef'e' cultivar, registered in Israel to HaZera company) in a controlled environment room at 25 ± 3°C and 14:10 L:D cycle, light intensity 6400 lum/ft² for over 50 generations prior to commencing experiments as previously described in Ben-Aziz et al . [ 47 ]. A colony of the fall army worms (FAW: Spodoptera frugiperda ) caterpillars was maintained on an artificial bean-based diet in a controlled environment room at 25 ± 3°C, approximately 60°C humidity, and 12:12 L:D cycle. Generating a stable bx6 mutant in a Triticum turgidum cv. Svevo wheat background The Bx6 knockout (ko) line was generated using the CRISPR–Cas9 genome editing system in Triticum turgidum as described by Hayta et al . [ 48 ]. Briefly, immature embryos were isolated, inoculated with Agrobacterium that holds target gene plasmid constructs, regenerated on Hygromycin B antibiotic, and acclimatized in soil ( Fig. S2 ). The genomic sequence of a target gene, TtBX6 (NCBI GeneBank accession: KY924305; IWGSC gene ID: TRITD2Bv1G015490), was retrieved from the Ensembl plant database ( https://plants.ensembl.org/index.html ). CRISPR guide RNAs were designed to target exons in the 5’ region of the gene using the CRISPOR software ( http://crispor.tefor.net/ ). The first and second top-ranked guide RNAs were selected based on their low probability of off-targets. Guide RNAs were assembled into the JD633 vector containing the Cas9 coding sequence and the growth-regulating factor GR4-GIF using Golden Gate assembly [ 49 ]. The construct was introduced into T. turgidum plants via agro-transformation. Transgenic plants were screened by PCR and Sanger sequencing to identify mutations at the target site ( Fig. S3 ). Lines carrying indel mutations resulting in loss of BX6 function were selected and used for further analyses ( Fig. S4 ). Salt stress Salt stress was imposed by irrigating wheat seedlings with 150 mM of sodium chloride (NaCl) starting four days after germination [ 50 ]. This salt concentration is commonly used for testing salt tolerance in wheat since it is considered a moderate-to-severe salt stress condition, simulating saline soil environments [ 51 – 53 ]. Importantly, this level of salinity stress was sufficient to induce BXD accumulation in the leaves and roots of SV WT plants ( Fig. S1 ). Irrigation with 150 mM of NaCl or water was done at two-day intervals. Tissues from leaves and roots were harvested ten days after salt stress was applied, and then the following parameters were measured. Chlorophyll content, relative water content, and biomass analysis Chlorophyll content, relative water content (RWC), and shoot biomass were measured as described previously [ 44 ]. Chlorophyll was extracted from fresh leaves in in ice-cold 80% acetone and centrifuged at 5,000 g for 5 minutes. Absorbance was recorded at 663 nm and 645 nm to quantify chlorophyll a and b, and total chlorophyll content was calculated according to Arnon (1949) [ 54 ] : $$\:Total\:cholophyll\:\left(\raisebox{1ex}{$mg$}\!\left/\:\!\raisebox{-1ex}{$g\:FW$}\right.\right)=\:\frac{(20.2\times\:{A}_{645})+(8.02\times\:{A}_{663})}{1000\times\:W}\:\text{X}\:V$$ where V is the extraction volume and W is the sample weight. RWC was determined from fresh weight (FW), turgid weight (TW), and dry weight (DW) of leaves or roots after rehydration and oven drying, using the following formula [ 55 ]: $$\:RWC\:\left(\%\right)=\frac{FW-DW}{TW-DW}\times\:100$$ Shoot biomass was assessed by measuring fresh and dry weights of aboveground tissues, and biomass percentage was calculated as below: $$\:Shoot\:biomass\:\left(\%\right)=\frac{FW-DW}{FW}\times\:100$$ Electrolyte leakages Electrolyte leakage was measured as described by Bajji et al . [ 56 ] to determine the membrane stability of salt-treated wheat leaves. The tissues were washed three times with deionized water to remove surface-adhered electrolytes. The leaflets were placed in a 50-mL Falcon tube containing 20 mL of DDW and shaken overnight at room temperature. First, the initial electrical conductivity (EC) was measured (C i ) and then autoclaved at 120°C for 20 min, and the maximum final EC (C m ) was obtained after equilibration at RT. The percentage of electrolyte leakage was defined as follows: $$\:\text{E}\text{l}\text{e}\text{c}\text{t}\text{r}\text{o}\text{l}\text{y}\text{t}\text{e}\:\text{l}\text{e}\text{a}\text{k}\text{a}\text{g}\text{e}\:\left(\text{%}\right)=\:\frac{Initial\:leakage\:\left({C}_{i}\right)}{Maximum\:leakage\:\left({C}_{m}\right)}\times\:100$$ Spectrophotometric assay for hydrogen peroxide Hydrogen peroxide (H 2 O 2 ) levels were measured using a spectrophotometric assay with minor modifications to the method described by Hao et al . [ 44 ]. Approximately 50 mg of leaf tissue was extracted in chilled potassium phosphate buffer (pH 7.5). The extract was incubated with a reaction mixture containing 4-aminoantipyrine, sodium 3,5-dichloro-2-hydroxybenzenesulfonate, and horseradish peroxidase. After 10 min, absorbance was measured at 500 nm, and H 2 O 2 concentrations were determined using a standard calibration curve. Aphid body weight and fecundity assays Aphid body weight measurement and fecundity assays were conducted following the method by Nalam et al. [ 57 ]. For body weight measurements, 10 adult R. padi aphids were confined to the second leaves of wheat plants for 96 h, using a clip-cage. Subsequently, the aphids were collected and weighed immediately to estimate body weight changes. Dry weights were obtained after drying the aphids overnight at 60°C. Six biological replicates were performed for each treatment in this experiment. For the aphid fecundity assay, three age-synchronized adult aphids were confined to a whole plant using breathable cellophane bags. The number of aphid progeny was counted after 96 h of feeding. Aphid feeding behavior analysis by Electrical Penetration Graph (EPG) The aphid feeding behavior was monitored on the second leaf of wheat plants using the EPG on a GIGA 8 complete system (EPG Systems, Wageningen, the Netherlands) [ 58 ]. The aphids used in this experiment were age-synchronized on wheat plants and starved for one hour before the recording. Two-week-old plants were used for analysis. An 18 µm diameter gold wire was attached to the dorsal surface of each adult R. padi abdomen using silver glue [ 59 ]. The plants were placed into a Faraday cage, electrodes were placed into the pots, and then the aphids were allowed to contact the leaf surface, and their probing was recorded. The aphids were allowed to feed for 8 h while the feeding behavior was recorded. The waveforms were digitized at 100 Hz with an analog-to-digital converter, and patterns were annotated as described previously [ 58 , 60 ]. A computer was connected to the Giga direct current amplifier, and the waveforms were collected every 30 s with the Stylet + d software (v01.30). EPG waveforms were analyzed using the Stylet + a software [ 57 , 61 ], and an Excel workbook was used to calculate the behavioral parameters automatically [ 62 ]. The feeding behavior of aphids was compared by analyzing the time spent in each of the four main phases: pathway phase (PP), non-probing phase (NP), sieve element phase (SEP), and xylem phase (G). The subphases within SEP that indicate phloem salivation (E1) and phloem ingestion (E2) were also analyzed. Parameters such as the time to 1st probe, the total number of probes, and the number of potential drops (PD), which indicate the aphid's health [ 63 ], were calculated. The potential E2 index, number of E1 and E2 waveforms, total time spent in E1 and E2, and percent time spent in E2 greater than 10 min indicate phloem acceptability and plant defense response [ 64 ]. The experiment was repeated until 15 replicates were obtained for each treatment. However, a recording was not considered a replicate if aphids spent more than 70% of the recording time in the non-probing, xylem, and derailed stylet phase. Thus, each treatment's final number of replicates differed (n = 14–19). The data were rank transformed, and differences between means were determined using the Wilcoxon test with Steel's method for nonparametric multiple comparisons with control JMP (SAS; www.jmp.com , USA) [ 65 , 66 ]. Two-spotted spider mite and caterpillar bioassays Ten adult TSSM females of mixed ages were placed on the abaxial side of a wheat leaf segment (4 cm long) and kept on 0.1% agar media in a plate under growth room conditions. Mortality and fecundity of the mites were recorded after 48 hours under a stereo-microscope [ 67 ]. In total, 10 leaf segments were tested for each genotype. For the caterpillar bioassay, three second-instar larvae of fall army worms (FAW; Spodoptera frugiperda ) were confined to the whole plant of 14-day-old SV WT or bx6-3 mutant plants using a breathable cellophane bag. The body weight of caterpillars was measured before and after two days of feeding (mg fresh weight). Then, the gained body weight was calculated using the following formula: $$\:\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{g}\text{a}\text{i}\text{n}\text{e}\text{d}\:\left(\%\right)=\left(\frac{Final\:weight-Initial\:weight}{Final\:weight}\right)\times\:100$$ Benzoxazinoid analysis using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) Benzoxazinoids (BXDs) were extracted and quantified following previously described methods [ 27 , 43 , 44 ]. Briefly, approximately 20 mg of tissue from the top-part of second leaves of pathogen-treated or stressed wheat plants was extracted in 80% methanol containing 0.1% formic acid (1:10, w/v) with benzoxazolin-2(3H)-one (BOA) as an internal standard. After centrifugation and filtration, samples were analyzed by HPLC using a C18 reverse-phase column with a water-acetonitrile gradient (both with 0.1% formic acid). BXDs were detected by UV-vis spectroscopy (190–400 nm) and quantified by comparison to authentic standards and calibration curves. Final metabolite levels were normalized to fresh weight and confirmed by retention time and UV spectra. Analysis of callose deposition Callose deposition was analyzed following established methods in Hao et al . [ 44 ]. In brief, leaf segments (~ 4 cm) from aphid-treated or control plants were decolorized in 80% acetone, washed with phosphate buffer (pH 9), and stained with 0.01% aniline blue. Samples were mounted and visualized under a fluorescence microscope using UV excitation. Multiple images per leaf were captured, and callose deposition was quantified by manually counting particles and normalizing to image area. Statistical analysis Statistical analysis, such as Student's paired t -test & Analysis of variance (ANOVA), was performed using Microsoft Excel and JMP Pro 14 (SAS; www.jmp.com , USA), respectively. Two-way ANOVA was performed using R v4.1.1. Data were tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene's test prior to performing statistical analyses. A Log 10 transformation was applied to the data before conducting the ANOVA to meet the assumptions of normality and homogeneity of variances. Statistical significance was considered when P values were less than 0.05 or 0.01, as indicated by one asterisk or two asterisks, respectively. Error bars represent standard errors of the mean. Results Herbivory and salinity stress elevated the levels of methoxylated BXDs in the leaves of wheat To investigate whether herbivory and salinity stress induce the accumulation of BXDs in tetraploid wheat, we analyzed the levels of BXDs in pest-damaged or salt-stressed leaves of SV WT plants using HPLC-DAD. As shown in Fig. 1 , the methoxylated forms of BXDs such as DIMBOA-Glc, DIMBOA, DIM 2 BOA-Glc, or HDMBOA-Glc, or HM 2 BOA-Glc were affected differently by the applied stresses. Aphid feeding and salt stress induced accumulation of DIMBOA-Glc in leaves, while DIMBOA and HM 2 BOA-Glc were increased under spider mite feeding. Caterpillar herbivory significantly elevated the levels of DIM 2 BOA-Glc, HDMBOA-Glc, and HM 2 BOA-Glc in the leaves. Additionally, we observed the effect of salt stress on the levels of BXDs in the roots of SV WT plants ( Supp. Figure 1 ). Silencing BX6 resulted in a complete loss of methoxylated BXDs To determine the role of BXDs in wheat herbivore resistance or salt tolerance, we generated a BXD -deficient mutant in the background of tetraploid wheat ( Triticum turgidum cv. Svevo) using the CRISPR-Cas9 system ( Fig. S2-4 ), by targeting the key biosynthetic gene BX6 , which encodes a dioxygenase catalyzing a central step in BXD biosynthesis [ 27 ]. The bx6 knock-out line displayed a clear BXD metabolic phenotype, characterized by the absence of methoxylated BXDs (DIMBOA, DIMBOA-Glc, HDMBOA-Glc, and DIM 2 BOA-Glc) in both leaf and root tissues, accompanied by a strong accumulation of the BX6 precursor DIBOA-Glc. Silencing BX6 enhanced performance of insect herbivores We first examined the defensive role of BXDs in tetraploid wheat by testing the susceptibility of BXD -deficient mutant plants to insects with different feeding types. First, we checked the feeding performance and behavior of a phloem-feeding insect ( R. padi ). The aphids performed better when fed on the bx6-3 mutant plants lacking methoxylated BXDs, as they had greater fecundity and body weight than the control (Fig. 2 A and B) . We also studied the feeding behavior of aphids in the phloem using an Electrical Penetration Graph (EPG) system. Although there was no difference in overall feeding behavior ( Fig. S5 and Table S1 ), the potential E2 index, phloem ingestion phase, was significantly higher in the aphids fed on bx6-3 mutants (Fig. 2 C). Next, we evaluated the survival and oviposition rates of a cell-content-feeding spider mite ( T. urticae ) on bx6 mutant plants. The mites showed higher survival rates and egg numbers laid when fed on the bx6-3 mutant plants than when fed on the WT plants (Fig. 2 D and E) . Lastly, we checked the feeding performance of a chewing insect (FAW; S. frugiperda ) on the mutant plants. Interestingly, the caterpillars' performance analysis (percentage weight gained) showed no difference between feeding on WT or mutant plants (Fig. 2 F). Taken together, the BX6- deficient mutant plants—lacking a key gene in BXD biosynthesis—were more susceptible to cell-sap suckers, while chewing insects feeding on them remained unaffected. This confirmed the defensive role for BXDs in wheat against certain types of herbivory. Silencing BX6 impairs callose deposition To investigate the regulatory role of benzoxazinoids in plant defense, we evaluated callose deposition in the leaves of SV WT or bx6-3 plants under aphid feeding. As shown in Fig. 3 , callose deposition was induced upon aphid feeding in both genotypes of the plants, with significantly lower levels observed in the mutant plants. Interestingly, the callose deposition was lower in the control leaves of bx6-3 plants compared to SV WT control leaves. Moreover, quantification of callose spots showed that the number of callose particles was higher in both control and aphid-treated leaves of SV WT compared to the control or aphid-treated leaves of bx6-3 plants, respectively. Silencing BX6 reduced plant tolerance to salt stress To test whether BXDs are involved in plant response to salt stress, wheat plants (SV WT and bx6-3 ) were irrigated with 150 mM NaCl. To assess the physiological and biochemical responses under salt stress, several parameters were measured in salt-stressed 14-day-old wheat plants. These included total chlorophyll, plant biomass, relative water content in the leaf and root, membrane stability, and reactive oxygen species (ROS), specifically hydrogen peroxide (H 2 O 2 ). As shown in Fig. 4 , all parameters measured were affected by either individual salt stress, genotype or the interaction of both these factors. Total chlorophyll content was significantly reduced in salt-stressed leaves of either WT or bx6-3 plants, with no significant interaction between genotype and salt treatment. Similarly, the shoot biomass was affected by salt stress or genotype, but no interaction effect was observed. Next, salt stress negatively impacted the water content in the leaf in both WT and mutant plants with no effect of genotype or interaction effect. In addition, the water content in the root was significantly lower in the salt-stressed mutant plants with a simple t -test, however; ANOVA showed no effect of either genotype or salt stress. While electrolyte leakage and H 2 O 2 levels increased in both WT and mutant leaves under salt stress, they were significantly affected by genotype, salt stress, and their interactions. The mutant plants exhibited a 1.1-fold higher electrolyte leakage and a 1.3-fold increase in H 2 O 2 levels (approximately 90 µmol/g FW higher) compared to the WT (Fig. 4 E-F). These results suggest that membrane integrity is more severely compromised in the mutant plants as they experienced greater oxidative stress under salt stress conditions. All in all, the BX6- deficient mutant plants were more susceptible to salt stress than WT plants. Discussion Due to the sessile nature of plants and the adverse effects of climate change, plants are constantly exposed to environmental biotic and abiotic stresses. Understanding the roles of specialized metabolites in plant response to abiotic and biotic stresses is crucial to comprehending their impact on (agro)ecosystem functioning and diversity. In this study, we investigate the potential role of benzoxazinoids in conferring salt tolerance and herbivore resistance in wheat. Adverse effects of salt stress on wheat plant physiology Salt stress poses a significant challenge to durum wheat production, particularly in arid and semi-arid regions where irrigation with poor drainage leads to salt accumulation. Salt stress negatively impacts plant physiology and biochemical processes, thereby impairing cellular mechanisms and metabolism. Exposure to this stress can cause both osmotic and oxidative stress in plants. Plants exposed to salt stress, typically ranging from 50 to 200 mM of NaCl, often exhibit a decrease in chlorophyll levels, which limits photosynthetic efficiency, thereby reducing overall biomass production [ 68 , 69 ]. The decrease in chlorophyll can be associated with oxidative stress caused by the accumulation of reactive oxygen species, which can lead to cell or chloroplast damage [ 70 ]. Moreover, salt stress critically affects water content in the leaves and roots, leading to wilting and reduced leaf expansion [ 71 ]. This stress also increases electrolyte leakage, indicating membrane damage, compromised cell integrity, and stress-induced injury [ 56 , 72 ]. In our experiments, we also observed the negative impact of salt stress on the physiology of wheat seedlings, regardless of genotype (Fig. 4 ). The critical effects of reduced chlorophyll content, reduced water content in leaves and roots, and increased levels of electrolyte leakage and ROS in response to the applied abiotic stress, emphasize the importance of understanding the physiological response of plants to develop breeding strategies that will improve abiotic stress tolerance [ 73 ]. Benzoxazinoids are involved in both herbivore resistance and salt tolerance in wheat Plant specialized metabolites are well recognized for their roles in defense against biotic stress, but they also contribute significantly to tolerance of abiotic stresses [ 74 – 77 ]. These metabolites, particularly DIMBOA-Glc, are abundant in young leaves and decrease as the plant develops [ 44 – 46 ], a pattern we also observed in our study ( Fig. S1 B ). Accordingly, all experiments were conducted on two-week-old wheat seedlings. BXDs have previously been reported to be induced by drought, salt and aphid stress [ 43 , 44 , 78 , 79 ]. Our study also demonstrates that BXD levels in the leaves and roots of tetraploid wheat are modulated by cell sap sucking herbivores and salt stress, highlighting their broader involvement in wheat stress responses. BXDs have been reported to exhibit antifeedant, antixenotic, antimicrobial, and allelopathic activities under biotic stresses [ 12 , 26 ], but their functions in plant responses to abiotic stresses remains are still largely unexplored. In maize, DIMBOA-Glc levels are negatively correlated with susceptibility to R. maidis aphids [ 39 ]. Aphid resistance in maize and wheat is associated with the abundance of DIMBOA and DIMBOA-Glc and the induction of callose deposition [ 26 , 35 , 80 ]. Callose deposition, triggered by aphid feeding, is a key phloem defense response that seals sieve pores to restrict nutrient flow to piercing-sucking arthropods [ 81 ]. Recently, we demonstrated that drought stress and aphid feeding enhance BXD accumulation and callose deposition in the leaves of hexaploid wheat seedlings, which correlated with reduced aphid performance [ 44 ]. Here, we provided evidence for the involvement of BXDs in callose deposition upon aphid feeding using a stable bx6 mutant (Fig. 3 ), thereby validating the role of BXDs in wheat defense against phloem feeding insects such as R. padi aphids and potentially other cell-sap-feeding insects. BXDs are also involved in plant defense against leaf-chewing insects [ 27 , 43 , 78 , 82 ]. However, the effect of BXDs on caterpillars of leaf-chewing insects may be species-specific. For example, Shavit et al. [ 27 ] reported that silencing of wheat BX6 in T. aestivum showed no change in the body weight of caterpillars of generalist S. littoralis . In contrast, another study showed better performance of the same caterpillar species on TaMYB31 -silenced T. aestivum plants [ 43 ]. In another report, Li et al. [ 26 ] showed that overexpression of ZmBX12 in T. aestivum did not affect the body weight of S. littoralis caterpillars, whereas the caterpillars showed preferred feeding and more significant consumption area on wild-type plants than BX12 -overexpressed plants. In our study, the performance of specialist S. frugiperda caterpillars was unaffected by BX6 -deficiency. Yet, we observed the accumulation of DIM 2 BOA-Glc, HDMBOA-Glc, and HM 2 BOA-Glc in leaves of SV WT plants upon S. frugiperda herbivory, which is in line with previous findings in maize that feeding of S. littoralis and S. frugiperda caterpillars on maize seedlings highly induced DIMBOA-Glc methylation and the production of HDM 2 BOA-Glc [ 14 ]. Taken together, as it was suggested by Li et al. [ 26 ], DIMBOA-Glc O -methylation increases the plant’s resistance to caterpillars through antixenosis but decreases resistance to aphids through antibiosis. Recent studies have shown that the two-spotted mite (TSSM, T. urticae ) causes damage to cereal crops such as maize, rice, barley, and wheat, making it a potentially significant pest for these crops [ 15 , 16 ]. TSSM feeding on BXD -deficient mutant plants, including BX1 , BX2 , and BX6 Ds transposon insertion mutants in maize or VIGS-silenced bx6 or TaMYB31 wheat plants have been shown to have an increased number of progenies [ 27 , 38 , 43 , 83 ]. We also observed higher survival rates and egg numbers laid by adult TSSM females when fed on the bx6-3 mutant plants than those fed on the WT plants (Fig. 2 ), suggesting that BXDs inhibit TSSM colonization. These findings highlight the potential role of BXDs in wheat defense against cell-content-feeding arthropods such as TSSM, warranting further investigation. The roles of plant specialized metabolites in abiotic stress tolerance remain largely unexplored, with only a limited number of studies investigating their involvement in plant responses to such stresses. For instance, a study showed that both flavonoid levels and the expression of key genes for flavonoid biosynthesis increased in Atriplex canescens plants under salt conditions. Notably, overexpression of chalcone isomerase, a key enzyme in flavonoid biosynthesis, in Arabidopsis reduced ROS accumulation and mitigated membrane damage, suggesting that flavonoids contribute to salt tolerance in A. canescens through ROS scavenging [ 84 ]. Similarly, a study by Batyrshina et al. [ 43 ] reported significant accumulation of BXDs in the leaves of hexaploid wheat seedlings germinated on salt solution, indicating a possible role in salt tolerance. This response was notably stronger than that induced by polyethylene glycol (PEG) and low-temperature treatments. In line with these findings, the present study demonstrates that BXD levels are modulated in both the leaves and roots of tetraploid wheat seedlings in response to salt stress (Fig. 1 & S1 ). Furthermore, BXD- deficient mutant plants lacking a key biosynthetic gene ( BX6 ) exhibited greater reductions in chlorophyll, biomass, and water content in the leaves and roots, along with increased electrolyte leakage and ROS accumulation under salt stress. These results suggest that elevated BXD levels under saline conditions may contribute to oxidative stress tolerance by enhancing the plant's ROS-scavenging capacity and protecting against lipid peroxidation and other forms of oxidative damage. However, further studies are required to fully elucidate the underlying mechanisms. It is important to note that, in contrast to Shavit et al . [ 27 ], who used VIGS to transiently suppress BX6 in hexaploid wheat ( T. aestivum ), this study employed stable CRISPR/Cas9 knockout lines generated on the background of tetraploid wheat ( T. turgidum ), allowing the assessment of a complete and heritable loss of function. Although both studies performed insect assays using the same species and observed comparable susceptibility outcomes, the use of stable mutants provides stronger genetic evidence for the role of BX6 in defense. Additionally, this work extends previous findings by examining the abiotic stress responses of the mutants: bx6 seedlings exhibited increased sensitivity to salt stress, offering functional insight not addressed in earlier studies. It is also worth noting that the accumulation of DIBOA-Glc in the bx6 mutant could contribute to the observed defense or stress responses. As reported by Tzin et al. [ 83 ], the effects of BXDs on R. maidis aphids and two-spotted spider mite progeny were stronger in bx1::Ds and bx2::Ds mutants than in bx6::Ds transposon insertion mutants in maize. This pattern suggests substantial functional redundancy in the BXD pathway; notably, the partially toxic DIBOA-Glc is still produced in the bx6 mutant, which may contribute to residual defense activity. Therefore, while the loss of methoxylated BXDs likely represents the primary driver of the bx6 phenotype, a contributory role of elevated DIBOA-Glc cannot be completely excluded. Overall, the combination of stable genome editing and expanded phenotypic characterization reveals new aspects of BX6 function in both abiotic and biotic stress responses. Conclusions In this study, we provide evidence for the involvement of BXDs in both T. turgidum wheat herbivore resistance and salt stress tolerance (Fig. 5 ). While adverse effects of salt stress were also observed in wild-type wheat, a more severe impact of the salt stress was found in the mutant plants lacking a key BXD-biosynthetic gene, BX6 . The bx6-3 plants also showed enhanced susceptibility to aphid and mite feeding. Overall, our results indicate that BXDs play an important role in wheat response to salt stress through ROS scavenging and the key enzyme gene BX6 in BXDs biosynthesis pathway of wheat has the potential to improve the stress resilience of cereal crops. Abbreviations BX Benzoxazinoid biosynthetic genes BXDs Benzoxazinoid metabolite compounds CRISPR Clustered, regularly interspaced short palindromic repeats DDW Double distilled water DIBOA 2,4-dihydroxy-1,4-benzoxazin-3-one DIBOA-Glc 2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one DIM 2 BOA-Glc 2,4-dihydroxy-7,8-dimethoxy-1,4-benzoxazin-3-one glucoside DIMBOA 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one DIMBOA-Glc 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside H 2 O 2 Hydrogen peroxide HDMBOA-Glc 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside HM 2 BOA-Glc 2-hydroxy-7,8-dimethoxy-2h-1,4-benzoxazin-3(4h)-one glucoside NaCl Sodium chloride OMT O -methyltransferase qRT-PCR Quantitative Real-time polymerase chain reaction R. padi Rhopalosiphum padi ROS Reactive oxygen species S. frugiperda Spodoptera frugiperda SV Svevo T. turgidum Triticum turgidum TSSM Two-spotted spider mite WT Wild-type Declarations Acknowledgements This research was funded by the German Research Foundation DFG (grant no. KO 4781/4-1) and the Israel Science Foundation (grant No. 329/20). We would like to thank Prof. Shimon Rachmilevitch for helping us with carrying bx6 seeds from Prof. Distelfeld’s lab to Prof. Tzin’s lab. Funding The German Research Foundation DFG (grant no. KO 4781/4-1) and the Israel Science Foundation (grant No. 329/20). Author information Authors and affiliations French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben Gurion, 8499000, Israel Let Kho Hao, Reut Shavit, Beery Yaakov, Vered Tzin Laboratory and Pilot Plant Manager, ALGIECEL Company, Copenhagen, Denmark Reut Shavit Department of Entomology, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel Adi Kliot, Murad Ghanim Department of Evolutionary and Environmental Biology, and Institute of Evolution, University of Haifa, Haifa, Israel Assaf Distelfeld Max Planck Institute for Chemical Ecology, Department of Natural Product Biosynthesis, D-07745, Jena, Germany Tobias G. Köllner Contribution L.K.H. and V.T. conceived and designed the study. L.K.H. developed the methodology, performed the experiments, and curated the data. R.S, L.K.H, and A.D developed stable bx6 mutants, contributed to manuscript review and editing. L.K.H. prepared the original draft of the manuscript. V.T. managed the project, provided resources, supervised the research, manuscript review, and editing. A.K and M.G. contributed to experiments with insects, manuscript review, and editing. T.G.K. provided resources and assisted with manuscript review and editing. B.Y. contributed to reviewing and editing the manuscript. All authors read, reviewed, and approved the final version of the manuscript. Corresponding author Correspondence to Dr. Beery Yaakov, [email protected] , +972544727025 . Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare no competing interests. References FAOSTAT. FAO Statistical Database: Food and Agriculture Organization of the United Nations. 2014. 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J Plant Sci Res. 2019;35:171–82. https://doi.org/10.32381/jpsr.2019.35.02.4. Jackson P, Robertson M, Cooper M, Hammer G. The role of physiological understanding in plant breeding; From a breeding perspective. F Crop Res. 1996;49:11–37. https://doi.org/10.1016/S0378-4290(96)01012-X. Yadav B, Jogawat A, Rahman MS, Narayan OP. Secondary metabolites in the drought stress tolerance of crop plants: A review. Gene Reports. 2021;23:101040. https://doi.org/10.1016/j.genrep.2021.101040. Davies KM, Albert NW, Zhou Y, Schwinn KE. Functions of flavonoid and betalain pigments in abiotic stress tolerance in plants. Annu Plant Rev Online. 2018;1:21–62. https://doi.org/10.1002/9781119312994.apr0604. Yang L, Wen KS, Ruan X, Zhao YX, Wei F, Wang Q. Response of plant secondary metabolites to environmental factors. Molecules. 2018;23:762. https://doi.org/10.3390/molecules23040762. Niculaes C, Abramov A, Hannemann L, Frey M. Plant protection by benzoxazinoids—recent insights into biosynthesis and function. Agronomy. 2018;8:143. https://doi.org/10.3390/agronomy8080143. Robert CAM, Mateo P. The chemical ecology of benzoxazinoids. Chimia (Aarau). 2022;76:928–38. https://doi.org/10.2533/chimia.2022.928. Sutour S, Doan VC, Mateo P, Züst T, Hartmann ER, Glauser G, et al. Isolation and structure determination of drought-induced multihexose benzoxazinoids from maize ( Zea mays ). J Agric Food Chem. 2024;72:3427–35. https://doi.org/10.1021/acs.jafc.3c09141. Betsiashvili M, Ahern KR, Jander G. Additive effects of two quantitative trait loci that confer Rhopalosiphum maidis (corn leaf aphid) resistance in maize inbred line Mo17. J Exp Bot. 2015;66:571–8. https://doi.org/10.1093/jxb/eru379. Will T, Van Bel AJE. Physical and chemical interactions between aphids and plants. In: Journal of Experimental Botany. Oxford Academic; 2006. p. 729–37. https://doi.org/10.1093/jxb/erj089. Wouters FC, Blanchette B, Gershenzon J, Vassão DG. Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores. Phytochemistry Reviews. 2016;15:1127–51. https://doi.org/10.1007/s11101-016-9481-1. Tzin V, Fernandez-Pozo N, Richter A, Schmelz EA, Schoettner M, Schäfer M, et al. Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays. Plant Physiol. 2015;169:1727–43. https://doi.org/10.1104/PP.15.01039. Feng S, Yao YT, Wang BB, Li YM, Li L, Bao AK. Flavonoids are involved in salt tolerance through ROS scavenging in the halophyte Atriplex canescens. Plant Cell Rep. 2024;43:1–17. https://doi.org/10.1007/s00299-023-03087-6. Additional Declarations No competing interests reported. Supplementary Files Supplementarytable.xlsx SuppsWheatBXDsInsectsSalt2026Feb.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 May, 2026 Reviews received at journal 29 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers invited by journal 10 Apr, 2026 Editor assigned by journal 10 Apr, 2026 Editor invited by journal 08 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 08 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9235518","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":621893434,"identity":"1126e0ce-f8db-4b06-989d-60064df0fb73","order_by":0,"name":"Let Kho Hao","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Let","middleName":"Kho","lastName":"Hao","suffix":""},{"id":621893435,"identity":"93a2e432-0458-4ab5-9f9c-b13264aab694","order_by":1,"name":"Reut Shavit","email":"","orcid":"","institution":"ALGIECEL Company","correspondingAuthor":false,"prefix":"","firstName":"Reut","middleName":"","lastName":"Shavit","suffix":""},{"id":621893436,"identity":"b3b1e1fd-b9a3-48f2-8b8b-23fa2e9b26f6","order_by":2,"name":"Beery Yaakov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBACCQbmhgMgBhvDwYYDH0AMdoJaGCFa+BkPNx6cAdLCTIQWMEOy+XjzYR4Qi5AWyfaDjYduVNyTMzh2sOGwza9t8nzMDIwfPubg1iLNk9hwOOdMsbHBGaCW3L7bhm3MDMySM7fh1iLHANSS25aQuOEGSEvPbUagFjZmXnxa+B8CVf4DarkPZFj23LYnqEVaAmRLQ0LizAagLQw/bicS1CI5A2h4zrEEY35gvBzsbbid3MbM2IzXLxLnkw9/zqlJkGNjOP74w48/t23ntzcf/PARjxZUwNgGJhuIVQ8Cf0hRPApGwSgYBSMFAADAb1715o/BhAAAAABJRU5ErkJggg==","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":true,"prefix":"","firstName":"Beery","middleName":"","lastName":"Yaakov","suffix":""},{"id":621893437,"identity":"c35fbf46-a0ff-4ab7-ab3e-8342247dbcef","order_by":3,"name":"Adi Kliot","email":"","orcid":"","institution":"Agricultural Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Adi","middleName":"","lastName":"Kliot","suffix":""},{"id":621893438,"identity":"b235281f-2a00-495e-b436-40d7ca91a1df","order_by":4,"name":"Murad Ghanim","email":"","orcid":"","institution":"Agricultural Research Organization","correspondingAuthor":false,"prefix":"","firstName":"Murad","middleName":"","lastName":"Ghanim","suffix":""},{"id":621893439,"identity":"49bd9065-2623-4eff-8103-78b22b578b87","order_by":5,"name":"Assaf Distelfeld","email":"","orcid":"","institution":"University of Haifa","correspondingAuthor":false,"prefix":"","firstName":"Assaf","middleName":"","lastName":"Distelfeld","suffix":""},{"id":621893440,"identity":"abacf40c-5fc7-4f38-8f65-764cbc4c08e7","order_by":6,"name":"Tobias G. Köllner","email":"","orcid":"","institution":"Max Planck Institute for Chemical Ecology","correspondingAuthor":false,"prefix":"","firstName":"Tobias","middleName":"G.","lastName":"Köllner","suffix":""},{"id":621893441,"identity":"7fa5a2dc-21cd-45fc-8337-461ec8fb89b9","order_by":7,"name":"Vered Tzin","email":"","orcid":"","institution":"Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Vered","middleName":"","lastName":"Tzin","suffix":""}],"badges":[],"createdAt":"2026-03-26 14:40:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9235518/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9235518/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107311082,"identity":"194d887d-4fb9-43bb-90b1-30a47cfcd13e","added_by":"auto","created_at":"2026-04-20 09:11:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1425461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of benzoxazinoid levels in the leaves of Svevo WT wheat plants under insect herbivory and salt stress by HPLC-DAD\u003c/strong\u003e. Tissues from pest-damaged (\u003cem\u003eRhopalosiphum padi, Tetranychus urticae\u003c/em\u003e, or \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) or salt-stressed (150 mM NaCl) leaves of 14-day-old wheat plants were extracted with methanol and BXDs were analyzed using HPLC-DAD. The amount of each BXD under each treatment was divided by its own control and presented in relative accumulation (fold-change). Student's \u003cem\u003et\u003c/em\u003e-test was performed to study the effect of pest feeding or salt stress on each compound in comparison to its specific control. Error bars represent standard errors of the mean (n = 5). Asterisks indicate significant differences, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 or * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 or n.s for not significant, whereas n.d. denotes not detected.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/1176f74a4f7229b9cc5673bc.png"},{"id":107311092,"identity":"92cb7cfa-7ec8-424a-b7a3-7a8659dac43b","added_by":"auto","created_at":"2026-04-20 09:11:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1336076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInsect bioassays on wheat \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebx6-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants\u003c/strong\u003e. A) Aphid fecundity: Aphids were age-synchronized, and two adults were confined to the 14-day-old wheat plants using breathable cellophane bags, and newborn nymphs were counted after 7 d (n = 10). B) Aphid body weight: Ten adult aphids were confined in a clip-cage to the top part of a second leaf of 14-day-old wheat plants for 96 h (n = 6) before being weighed. C) Aphid feeding behavior in the phloem: The dorsal surface of each adult \u003cem\u003eR. padi\u003c/em\u003e abdomen was attached with 18 μm diameter gold wire using silver glue for recording its feeding behavior by EPG system (n = 16 for SV WT and 19 for \u003cem\u003ebx6-3\u003c/em\u003e). The Wilcoxon test was used for evaluating statistical differences, determined by * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. D) Survival and E) oviposition of two-spotted spider mites (\u003cem\u003eT. urticae\u003c/em\u003e): Ten adult females were placed on the abaxial side of a segment of second leaf of 14-day-old plants for 48 h before mortality and the number of eggs were counted and the number of eggs per live female was calculated (n = 10). F) Caterpillar performance: Three second-instars FAW (\u003cem\u003eS. frugiperda\u003c/em\u003e) larvae were confined to the whole plant of 14-day-old SV WT or \u003cem\u003ebx6-3\u003c/em\u003e mutant plants with breathable cellophane bags for 48 h (n = 10).\u003cstrong\u003e \u003c/strong\u003eError bars represent standard errors of the mean. Asterisks indicate significant differences, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, or n.s for not significant, Student's \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/76d10895c6f5bfbe9b85281e.png"},{"id":107311270,"identity":"f96c1985-696c-4a60-84ac-38aff1de8940","added_by":"auto","created_at":"2026-04-20 09:12:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4476713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of callose depositions in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebx6-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e wheat leaves under aphid feeding.\u003c/strong\u003eLeaves of SV WT or \u003cem\u003ebx6-3\u003c/em\u003e wheat plans were infested with ten adult \u003cem\u003eR. padi \u003c/em\u003eaphids for 96 h and visualized under a microscope. A) Microscopic image of wheat leaf grown under aphid feeding. (B) Quantification of callose depositions in aphid-treated leaves. At least 15 images were taken from each biological replicate and the average number of callose deposits was calculated for each condition. Student \u003cem\u003et\u003c/em\u003e-test was performed to study the difference of callose deposits in \u003cem\u003ebx6-3\u003c/em\u003e leaves compared to SV WT. Asterisks indicate statistically significant difference, * \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05 \u0026amp; ** \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.01. Error bars indicate standard errors of the mean (n=6).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/573ef6e799985ae44006d500.png"},{"id":107311094,"identity":"a9c8a5c9-af36-4cf5-b4fd-1d7c95460d68","added_by":"auto","created_at":"2026-04-20 09:11:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2115640,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological and biochemical response of wheat \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebx6-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant plants to salt stress\u003c/strong\u003e. A) Total chlorophyll, B) Biomass, C) Leaf water content, D) Root water content, E) Membrane stability, F) Hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels, and G) FDR-adjusted \u003cem\u003eP\u003c/em\u003e-values of two-way ANOVA. Leaf tissues were collected from 14-day-old plants subjected to salt stress (150 mM NaCl) or control conditions. Student’s \u003cem\u003et\u003c/em\u003e-test was used to evaluate the effect of salt stress within each genotype. Asterisks indicate statistically significant differences: \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*), or \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (**). Two-way ANOVA was performed to assess the effects of salt treatment, genotype, and their interaction. In panel G, \u003cem\u003eP\u003c/em\u003e-values in bold indicate significant effects. Error bars represent the standard error of the mean (n = 5).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/21f697f4eb4bfbb93218f41a.png"},{"id":107311076,"identity":"183f3b51-31a6-45d7-915d-ec06d0e469ab","added_by":"auto","created_at":"2026-04-20 09:11:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4096925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model for the roles of benzoxazinoids in wheat herbivore resistance and salt tolerance.\u003c/strong\u003e Both herbivory and salt stress induce the accumulation of BXDs in \u003cem\u003eTriticum turgidum\u003c/em\u003e wheat plants. In herbivore resistance, BXDs contribute through antixenosis (non-preference) and antibiosis (toxicity). In salt stress, BXDs may aid in maintaining ROS homeostasis, thereby enhancing stress tolerance.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/584ba9e813e2152cbef895ec.png"},{"id":107311619,"identity":"ad532af3-d6dc-4a39-b228-f134e2a456d1","added_by":"auto","created_at":"2026-04-20 09:13:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13417771,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/b3dd38dc-abf1-465e-865c-07acd391c00d.pdf"},{"id":107311141,"identity":"e8aeea90-8b47-43cc-911b-13c120b39eaa","added_by":"auto","created_at":"2026-04-20 09:11:55","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16316,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/5124098e12eb7cf9bac43bd0.xlsx"},{"id":107311054,"identity":"dc5f36ff-aef0-4bfc-8ce2-b803a3b478fb","added_by":"auto","created_at":"2026-04-20 09:11:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3560416,"visible":true,"origin":"","legend":"","description":"","filename":"SuppsWheatBXDsInsectsSalt2026Feb.docx","url":"https://assets-eu.researchsquare.com/files/rs-9235518/v1/8fd6af326dbf564454c1569f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"BX6-dependent benzoxazinoid biosynthesis enhances herbivore resistance and salt stress tolerance in durum wheat Triticum turgidum","fulltext":[{"header":"Key message","content":"\u003cp\u003eFunctional loss of BX6 reveals benzoxazinoids as crucial mediators of wheat herbivore resistance and salt tolerance, linking specialized metabolism to environmental resilience.\u003c/p\u003e"},{"header":"Background","content":"\u003cp\u003eWheat (\u003cem\u003eTriticum\u003c/em\u003e spp.) is an economically and nutritionally important cereal crop, which occupies around 17% of the global cultivated area. Moreover, it provides food for about 35% of the world's population and approximately 20% of humanity's consumed calories [\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWheat production is adversely affected by both environmental stress and biotic stress. Salinity is one of the most severe environmental stresses, significantly reducing crop yield and quality worldwide. It is estimated that over 20% of cultivated land—responsible for producing one-third of the world's food—is affected by soil salinity, reducing productivity [\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e]. Salt stress has a strong effect on plant growth and development. Salinity disrupts cellular ultrastructure, impairs the photosynthetic apparatus, damages membranes, elevates reactive oxygen species (ROS) production, and reduces enzymatic activity, collectively limiting crop growth and yield [\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiotic stress due to herbivore pest infestation significantly reduces food quality and crop yield [\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e]. Aphids (order Hemiptera, family Aphididae) are major pests of wheat and other grass family species [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e]. Aphids consume water and nutrients from the phloem sap of plants, which poses both osmotic and chemical challenges due to its high solute concentration and defensive metabolites [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e]. Aphid feeding on plants causes substantial yield losses through direct damage and indirectly, through facilitation of secondary infections. Furthermore, aphids transmit nearly 40% of all known plant viruses, including several of the most destructive viral diseases in wheat, such as the wheat yellow mosaic virus and wheat streak mosaic virus [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOther notable pests of wheat include leaf-chewing insects such as caterpillars of the Lepidoptera order. Caterpillar infestation triggers extensive transcriptomic reprogramming in plants and production of specialized defensive metabolites [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile plant responses to aphids and caterpillars in grass crops are relatively well studied, the impact of mite herbivory is less so, despite them being prominent wheat pests, especially in warm climates. However, recent studies have shown that the two-spotted spider mite (TSSM; \u003cem\u003eTetranychus urticae\u003c/em\u003e) can significantly damage cereal crops, including maize, rice, barley, and wheat, highlighting its emerging importance as a pest [\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbiotic and biotic stresses, which negatively impact crop production, are expected to increase due to global climate change [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]. To overcome challenges imposed by these stressors, plants have evolved arrays of mechanisms, including specialized metabolite production.\u003c/p\u003e \u003cp\u003ePlant specialized metabolites (PSMs), also known as secondary metabolites, are diverse chemical compounds that play vital roles in plant responses to various stresses. PSMs were shown to function more in plant defense rather than in essential processes like growth and reproduction [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e], with over 200,000 known PSMs produced by plants [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Perhaps the most known function of PSMs is acting as antifeedants against arthropod herbivores; targeting the pests' nervous, digestive, and endocrine systems [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. PSMs also play several key roles in abiotic stress tolerance, such as antioxidant defense, membrane stabilization, metal chelation, and UV protection [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBenzoxazinoids (BXDs) are indole-derived specialized PSMs abundantly produced in Poaceae crops such as maize (\u003cem\u003eZea mays\u003c/em\u003e), wheat (\u003cem\u003eTriticum\u003c/em\u003e spp.), and rye (\u003cem\u003eSecale cereale\u003c/em\u003e) [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. BXDs' biosynthesis begins in the plastids with indole-3-glycerol phosphate lyase benzoxazinoneless 1 (BX1), converting indole-3-glycerol phosphate into indole, followed by cytochrome P450 enzymes (BX2-5), forming the core compound 2,4-dhydroxy-1,4-benzoxazin-3-one (DIBOA). DIBOA is stabilized through glycosylation by BX8 and BX9 and stored as DIBOA-Glc in the vacuole to prevent autotoxicity [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. Further modifications, by tailoring enzymes such as BX6 (a dioxygenase) and BX7 (an \u003cem\u003eO\u003c/em\u003e-methyltransferase), convert DIBOA-Glc into DIMBOA-Glc, the most abundant form of BXDs in wheat and maize [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Additional derivatives such as HDMBOA-Glc, TRIMBOA-Glc, DIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc, and HDM\u003csub\u003e2\u003c/sub\u003eBOA-Glc are produced through sequential tailoring enzymatic steps by additional \u003cem\u003eO\u003c/em\u003e-methyltransferases (BX10-12 \u0026amp; 14) and dioxygenase (BX13). To date, genes encoding BX6 and BX10 are the only tailoring enzyme genes that have been identified and characterized in wheat [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e], whereas those corresponding to BX7, BX11, BX12, BX13, and BX14 have not yet been discovered.\u003c/p\u003e \u003cp\u003eOwing to their abundance and essential roles in economically and nutritionally important crops, BXDs have been extensively studied for over 50 years [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. BXDs are primarily known for defense against various biotic stresses, including insect herbivores, microbial pathogens, and competing plant species [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. BXDs have been reported to affect the fitness of phloem feeding insects such as aphids [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e], chewing herbivores such as caterpillars [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e], cell content feeders such as spider mites [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e], and pathogens such as fungi [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Shavit \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] reported that silencing the wheat dioxygenase, \u003cem\u003eTaBX6\u003c/em\u003e, using virus-induced gene silencing (VIGS) increased wheat susceptibility to both aphids and spider mites, while caterpillar performance was unaffected. This indicates that \u003cem\u003eTaBX6\u003c/em\u003e plays a role in plant defense against sucking herbivores. Besides acting as direct defense compounds (toxins), BXDs, particularly DIMBOA-Glc or its aglucon DIMBOA, also function as regulators for other defensive processes, such as callose deposition [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChanges in the levels of BXDs upon environmental stresses have also been reported. For example, drought stress increases the levels of DIMBOA and DIBOA in maize [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e], and the \u003cem\u003eO\u003c/em\u003e-methyltransferase ZmBX12, which converts DIMBOA-Glc into HDMBOA-Glc, has been linked to drought adaptation in this plant [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition, Poschenrieder \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e] demonstrated that adding DIMBOA to the growth medium of an aluminum-susceptible maize line is sufficient to protect the plants from the adverse effects of aluminum, suggesting its potential role in safeguarding maize plants against aluminum toxicity. In wheat, Batyrshina \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e] reported that environmental stresses such as drought, salinity, and cold affected the levels of BXDs in the leaves of wheat seedlings. These findings highlight a potential, unexplored role for BXDs in abiotic stress tolerance.\u003c/p\u003e \u003cp\u003eRecently, we demonstrated that drought triggers BXD accumulation and callose deposition in the leaves, which disrupt aphid feeding, supporting a dual role for BXDs in hexaploid wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) drought tolerance and aphid resistance [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Here, we investigate how wheat modulates BXD production in response to herbivory or salt stress and examined their roles in conferring herbivory and salt tolerance resistance using tetraploid wheat (\u003cem\u003eTriticum turgidum\u003c/em\u003e). Understanding how biotic and abiotic stresses alter BXD profiles and how these changes contribute to plants' stress tolerance is crucial for developing sustainable approaches to address upcoming agricultural challenges.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003ePlant material, insect colony, and growth room conditions\u003c/h2\u003e\u003cp\u003eSeeds of \u003cem\u003eTriticum turgidum\u003c/em\u003e ssp. \u003cem\u003edurum\u003c/em\u003e cultivar Svevo (SV) or \u003cem\u003eBXD-deficient\u003c/em\u003e mutants were germinated on moistened Whatman filter paper. SV wheat seeds were obtained from laboratory of Prof. Assaf Distelfeld (Haifa University, Isarel), while \u003cem\u003eBXD-deficient\u003c/em\u003e mutants were generated in the laboratory of Prof. Vered Tzin (Ben-Gurion University of Negev, Israel). Two days after sowing, transplanted into individual pots containing a tuff-vermiculite mixture (2:1) supplemented with N-P-K fertilizer (20–20–20). Plants were grown in a controlled growth room under 16 h light/8 h dark cycle; 250–350 µmol photons m\u003csup\u003e− 2\u003c/sup\u003e s\u003csup\u003e− 1\u003c/sup\u003e light intensity from a 3000-lm LED, 22 ± 3°C; and 60 ± 10% relative humidity. Soil moisture was maintained by watering all pots equally for two days after transplanting [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. As BXDs are abundant in young wheat leaves but decline during plant development [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e], all experiments were performed on 14-day-old seedlings under the same growth room conditions unless otherwise stated.\u003c/p\u003e\u003cp\u003eA colony of bird cherry-oat aphids (\u003cem\u003eRhopalosiphum padi\u003c/em\u003e) was maintained on bread wheat seedlings (\u003cem\u003eT. aestivum\u003c/em\u003e cv. Rotem) as previously described [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e]. For all aphid bioassays, approximately two-week-old apterous adults were used.\u003c/p\u003e\u003cp\u003eA colony of Two- Spotted Spider Mites (TSSM, \u003cem\u003eTetranychus urticae\u003c/em\u003e) were reared on \u003cem\u003eCapsicum annum\u003c/em\u003e plants (commercial line 'Ef'e' cultivar, registered in Israel to HaZera company) in a controlled environment room at 25 ± 3°C and 14:10 L:D cycle, light intensity 6400 lum/ft² for over 50 generations prior to commencing experiments as previously described in Ben-Aziz \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA colony of the fall army worms (FAW: \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) caterpillars was maintained on an artificial bean-based diet in a controlled environment room at 25 ± 3°C, approximately 60°C humidity, and 12:12 L:D cycle.\u003c/p\u003e\u003cp\u003e \u003cb\u003eGenerating a stable\u003c/b\u003e \u003cb\u003ebx6\u003c/b\u003e \u003cb\u003emutant in a\u003c/b\u003e \u003cb\u003eTriticum turgidum\u003c/b\u003e \u003cb\u003ecv. Svevo wheat background\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eBx6\u003c/em\u003e knockout (ko) line was generated using the CRISPR–Cas9 genome editing system in \u003cem\u003eTriticum turgidum\u003c/em\u003e as described by Hayta \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. Briefly, immature embryos were isolated, inoculated with Agrobacterium that holds target gene plasmid constructs, regenerated on Hygromycin B antibiotic, and acclimatized in soil (\u003cb\u003eFig. S2\u003c/b\u003e). The genomic sequence of a target gene, \u003cem\u003eTtBX6\u003c/em\u003e (NCBI GeneBank accession: KY924305; IWGSC gene ID: TRITD2Bv1G015490), was retrieved from the Ensembl plant database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). CRISPR guide RNAs were designed to target exons in the 5’ region of the gene using the CRISPOR software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispor.tefor.net/\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The first and second top-ranked guide RNAs were selected based on their low probability of off-targets. Guide RNAs were assembled into the JD633 vector containing the Cas9 coding sequence and the growth-regulating factor GR4-GIF using Golden Gate assembly [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. The construct was introduced into \u003cem\u003eT. turgidum\u003c/em\u003e plants \u003cem\u003evia\u003c/em\u003e agro-transformation. Transgenic plants were screened by PCR and Sanger sequencing to identify mutations at the target site (\u003cb\u003eFig. S3\u003c/b\u003e). Lines carrying indel mutations resulting in loss of \u003cem\u003eBX6\u003c/em\u003e function were selected and used for further analyses (\u003cb\u003eFig. S4\u003c/b\u003e).\u003c/p\u003e\u003ch3\u003eSalt stress\u003c/h3\u003e\u003cp\u003eSalt stress was imposed by irrigating wheat seedlings with 150 mM of sodium chloride (NaCl) starting four days after germination [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. This salt concentration is commonly used for testing salt tolerance in wheat since it is considered a moderate-to-severe salt stress condition, simulating saline soil environments [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Importantly, this level of salinity stress was sufficient to induce BXD accumulation in the leaves and roots of SV WT plants (\u003cb\u003eFig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Irrigation with 150 mM of NaCl or water was done at two-day intervals. Tissues from leaves and roots were harvested ten days after salt stress was applied, and then the following parameters were measured.\u003c/p\u003e\u003ch3\u003eChlorophyll content, relative water content, and biomass analysis\u003c/h3\u003e\u003cp\u003eChlorophyll content, relative water content (RWC), and shoot biomass were measured as described previously [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Chlorophyll was extracted from fresh leaves in in ice-cold 80% acetone and centrifuged at 5,000 g for 5 minutes. Absorbance was recorded at 663 nm and 645 nm to quantify chlorophyll a and b, and total chlorophyll content was calculated according to Arnon (1949) [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e] :\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Total\\:cholophyll\\:\\left(\\raisebox{1ex}{$mg$}\\!\\left/\\:\\!\\raisebox{-1ex}{$g\\:FW$}\\right.\\right)=\\:\\frac{(20.2\\times\\:{A}_{645})+(8.02\\times\\:{A}_{663})}{1000\\times\\:W}\\:\\text{X}\\:V$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere \u003cem\u003eV\u003c/em\u003e is the extraction volume and \u003cem\u003eW\u003c/em\u003e is the sample weight.\u003c/p\u003e\u003cp\u003eRWC was determined from fresh weight (FW), turgid weight (TW), and dry weight (DW) of leaves or roots after rehydration and oven drying, using the following formula [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]:\u003c/p\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:RWC\\:\\left(\\%\\right)=\\frac{FW-DW}{TW-DW}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003eShoot biomass was assessed by measuring fresh and dry weights of aboveground tissues, and biomass percentage was calculated as below:\u003c/p\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\:Shoot\\:biomass\\:\\left(\\%\\right)=\\frac{FW-DW}{FW}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003ch3\u003eElectrolyte leakages\u003c/h3\u003e\u003cp\u003eElectrolyte leakage was measured as described by Bajji \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e] to determine the membrane stability of salt-treated wheat leaves. The tissues were washed three times with deionized water to remove surface-adhered electrolytes. The leaflets were placed in a 50-mL Falcon tube containing 20 mL of DDW and shaken overnight at room temperature. First, the initial electrical conductivity (EC) was measured (C\u003csub\u003ei\u003c/sub\u003e) and then autoclaved at 120°C for 20 min, and the maximum final EC (C\u003csub\u003em\u003c/sub\u003e) was obtained after equilibration at RT. The percentage of electrolyte leakage was defined as follows:\u003c/p\u003e\u003cdiv id=\"Equd\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equd\" name=\"EquationSource\"\u003e\n$$\\:\\text{E}\\text{l}\\text{e}\\text{c}\\text{t}\\text{r}\\text{o}\\text{l}\\text{y}\\text{t}\\text{e}\\:\\text{l}\\text{e}\\text{a}\\text{k}\\text{a}\\text{g}\\text{e}\\:\\left(\\text{%}\\right)=\\:\\frac{Initial\\:leakage\\:\\left({C}_{i}\\right)}{Maximum\\:leakage\\:\\left({C}_{m}\\right)}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003ch3\u003eSpectrophotometric assay for hydrogen peroxide\u003c/h3\u003e\u003cp\u003eHydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels were measured using a spectrophotometric assay with minor modifications to the method described by Hao \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Approximately 50 mg of leaf tissue was extracted in chilled potassium phosphate buffer (pH 7.5). The extract was incubated with a reaction mixture containing 4-aminoantipyrine, sodium 3,5-dichloro-2-hydroxybenzenesulfonate, and horseradish peroxidase. After 10 min, absorbance was measured at 500 nm, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentrations were determined using a standard calibration curve.\u003c/p\u003e\u003ch2\u003eAphid body weight and fecundity assays\u003c/h2\u003e\u003cp\u003eAphid body weight measurement and fecundity assays were conducted following the method by Nalam \u003cem\u003eet al.\u003c/em\u003e [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e]. For body weight measurements, 10 adult \u003cem\u003eR. padi\u003c/em\u003e aphids were confined to the second leaves of wheat plants for 96 h, using a clip-cage. Subsequently, the aphids were collected and weighed immediately to estimate body weight changes. Dry weights were obtained after drying the aphids overnight at 60°C. Six biological replicates were performed for each treatment in this experiment. For the aphid fecundity assay, three age-synchronized adult aphids were confined to a whole plant using breathable cellophane bags. The number of aphid progeny was counted after 96 h of feeding.\u003c/p\u003e\u003ch3\u003eAphid feeding behavior analysis by Electrical Penetration Graph (EPG)\u003c/h3\u003e\u003cp\u003eThe aphid feeding behavior was monitored on the second leaf of wheat plants using the EPG on a GIGA 8 complete system (EPG Systems, Wageningen, the Netherlands) [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e]. The aphids used in this experiment were age-synchronized on wheat plants and starved for one hour before the recording. Two-week-old plants were used for analysis. An 18 µm diameter gold wire was attached to the dorsal surface of each adult \u003cem\u003eR. padi\u003c/em\u003e abdomen using silver glue [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. The plants were placed into a Faraday cage, electrodes were placed into the pots, and then the aphids were allowed to contact the leaf surface, and their probing was recorded. The aphids were allowed to feed for 8 h while the feeding behavior was recorded. The waveforms were digitized at 100 Hz with an analog-to-digital converter, and patterns were annotated as described previously [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. A computer was connected to the Giga direct current amplifier, and the waveforms were collected every 30 s with the Stylet\u003csup\u003e+\u003c/sup\u003ed software (v01.30). EPG waveforms were analyzed using the Stylet\u003csup\u003e+\u003c/sup\u003ea software [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e], and an Excel workbook was used to calculate the behavioral parameters automatically [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. The feeding behavior of aphids was compared by analyzing the time spent in each of the four main phases: pathway phase (PP), non-probing phase (NP), sieve element phase (SEP), and xylem phase (G). The subphases within SEP that indicate phloem salivation (E1) and phloem ingestion (E2) were also analyzed. Parameters such as the time to 1st probe, the total number of probes, and the number of potential drops (PD), which indicate the aphid's health [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e], were calculated. The potential E2 index, number of E1 and E2 waveforms, total time spent in E1 and E2, and percent time spent in E2 greater than 10 min indicate phloem acceptability and plant defense response [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. The experiment was repeated until 15 replicates were obtained for each treatment. However, a recording was not considered a replicate if aphids spent more than 70% of the recording time in the non-probing, xylem, and derailed stylet phase. Thus, each treatment's final number of replicates differed (n = 14–19). The data were rank transformed, and differences between means were determined using the Wilcoxon test with Steel's method for nonparametric multiple comparisons with control JMP (SAS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.jmp.com\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, USA) [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e].\u003c/p\u003e\u003ch3\u003eTwo-spotted spider mite and caterpillar bioassays\u003c/h3\u003e\u003cp\u003eTen adult TSSM females of mixed ages were placed on the abaxial side of a wheat leaf segment (4 cm long) and kept on 0.1% agar media in a plate under growth room conditions. Mortality and fecundity of the mites were recorded after 48 hours under a stereo-microscope [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. In total, 10 leaf segments were tested for each genotype.\u003c/p\u003e\u003cp\u003eFor the caterpillar bioassay, three second-instar larvae of fall army worms (FAW; \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e) were confined to the whole plant of 14-day-old SV WT or \u003cem\u003ebx6-3\u003c/em\u003e mutant plants using a breathable cellophane bag. The body weight of caterpillars was measured before and after two days of feeding (mg fresh weight). Then, the gained body weight was calculated using the following formula:\u003c/p\u003e\u003cdiv id=\"Eque\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Eque\" name=\"EquationSource\"\u003e\n$$\\:\\text{W}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t}\\:\\text{g}\\text{a}\\text{i}\\text{n}\\text{e}\\text{d}\\:\\left(\\%\\right)=\\left(\\frac{Final\\:weight-Initial\\:weight}{Final\\:weight}\\right)\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003ch2\u003eBenzoxazinoid analysis using high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD)\u003c/h2\u003e\u003cp\u003eBenzoxazinoids (BXDs) were extracted and quantified following previously described methods [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. Briefly, approximately 20 mg of tissue from the top-part of second leaves of pathogen-treated or stressed wheat plants was extracted in 80% methanol containing 0.1% formic acid (1:10, w/v) with benzoxazolin-2(3H)-one (BOA) as an internal standard. After centrifugation and filtration, samples were analyzed by HPLC using a C18 reverse-phase column with a water-acetonitrile gradient (both with 0.1% formic acid). BXDs were detected by UV-vis spectroscopy (190–400 nm) and quantified by comparison to authentic standards and calibration curves. Final metabolite levels were normalized to fresh weight and confirmed by retention time and UV spectra.\u003c/p\u003e\u003ch2\u003eAnalysis of callose deposition\u003c/h2\u003e\u003cp\u003eCallose deposition was analyzed following established methods in Hao \u003cem\u003eet al\u003c/em\u003e. [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. In brief, leaf segments (~ 4 cm) from aphid-treated or control plants were decolorized in 80% acetone, washed with phosphate buffer (pH 9), and stained with 0.01% aniline blue. Samples were mounted and visualized under a fluorescence microscope using UV excitation. Multiple images per leaf were captured, and callose deposition was quantified by manually counting particles and normalizing to image area.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis, such as Student's paired \u003cem\u003et\u003c/em\u003e-test \u0026amp; Analysis of variance (ANOVA), was performed using Microsoft Excel and JMP Pro 14 (SAS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.jmp.com\u003c/span\u003e\u003cspan class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, USA), respectively. Two-way ANOVA was performed using R v4.1.1. Data were tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene's test prior to performing statistical analyses. A Log\u003csub\u003e10\u003c/sub\u003e transformation was applied to the data before conducting the ANOVA to meet the assumptions of normality and homogeneity of variances. Statistical significance was considered when \u003cem\u003eP\u003c/em\u003e values were less than 0.05 or 0.01, as indicated by one asterisk or two asterisks, respectively. Error bars represent standard errors of the mean.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHerbivory and salinity stress elevated the levels of methoxylated BXDs in the leaves of wheat\u003c/h2\u003e \u003cp\u003eTo investigate whether herbivory and salinity stress induce the accumulation of BXDs in tetraploid wheat, we analyzed the levels of BXDs in pest-damaged or salt-stressed leaves of SV WT plants using HPLC-DAD. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the methoxylated forms of BXDs such as DIMBOA-Glc, DIMBOA, DIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc, or HDMBOA-Glc, or HM\u003csub\u003e2\u003c/sub\u003eBOA-Glc were affected differently by the applied stresses. Aphid feeding and salt stress induced accumulation of DIMBOA-Glc in leaves, while DIMBOA and HM\u003csub\u003e2\u003c/sub\u003eBOA-Glc were increased under spider mite feeding. Caterpillar herbivory significantly elevated the levels of DIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc, HDMBOA-Glc, and HM\u003csub\u003e2\u003c/sub\u003eBOA-Glc in the leaves. Additionally, we observed the effect of salt stress on the levels of BXDs in the roots of SV WT plants (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing\u003c/b\u003e \u003cb\u003eBX6\u003c/b\u003e \u003cb\u003eresulted in a complete loss of methoxylated BXDs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the role of BXDs in wheat herbivore resistance or salt tolerance, we generated a \u003cem\u003eBXD\u003c/em\u003e-deficient mutant in the background of tetraploid wheat (\u003cem\u003eTriticum turgidum\u003c/em\u003e cv. Svevo) using the CRISPR-Cas9 system (\u003cb\u003eFig. S2-4\u003c/b\u003e), by targeting the key biosynthetic gene \u003cem\u003eBX6\u003c/em\u003e, which encodes a dioxygenase catalyzing a central step in BXD biosynthesis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The \u003cem\u003ebx6\u003c/em\u003e knock-out line displayed a clear BXD metabolic phenotype, characterized by the absence of methoxylated BXDs (DIMBOA, DIMBOA-Glc, HDMBOA-Glc, and DIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc) in both leaf and root tissues, accompanied by a strong accumulation of the BX6 precursor DIBOA-Glc.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing\u003c/b\u003e \u003cb\u003eBX6\u003c/b\u003e \u003cb\u003eenhanced performance of insect herbivores\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe first examined the defensive role of BXDs in tetraploid wheat by testing the susceptibility of \u003cem\u003eBXD\u003c/em\u003e-deficient mutant plants to insects with different feeding types. First, we checked the feeding performance and behavior of a phloem-feeding insect (\u003cem\u003eR. padi\u003c/em\u003e). The aphids performed better when fed on the \u003cem\u003ebx6-3\u003c/em\u003e mutant plants lacking methoxylated BXDs, as they had greater fecundity and body weight than the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u003cb\u003eand B)\u003c/b\u003e. We also studied the feeding behavior of aphids in the phloem using an Electrical Penetration Graph (EPG) system. Although there was no difference in overall feeding behavior (\u003cb\u003eFig. S5 and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), the potential E2 index, phloem ingestion phase, was significantly higher in the aphids fed on \u003cem\u003ebx6-3\u003c/em\u003e mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Next, we evaluated the survival and oviposition rates of a cell-content-feeding spider mite (\u003cem\u003eT. urticae\u003c/em\u003e) on \u003cem\u003ebx6\u003c/em\u003e mutant plants. The mites showed higher survival rates and egg numbers laid when fed on the \u003cem\u003ebx6-3\u003c/em\u003e mutant plants than when fed on the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD \u003cb\u003eand E)\u003c/b\u003e. Lastly, we checked the feeding performance of a chewing insect (FAW; \u003cem\u003eS. frugiperda\u003c/em\u003e) on the mutant plants. Interestingly, the caterpillars' performance analysis (percentage weight gained) showed no difference between feeding on WT or mutant plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Taken together, the \u003cem\u003eBX6-\u003c/em\u003edeficient mutant plants\u0026mdash;lacking a key gene in BXD biosynthesis\u0026mdash;were more susceptible to cell-sap suckers, while chewing insects feeding on them remained unaffected. This confirmed the defensive role for BXDs in wheat against certain types of herbivory.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing\u003c/b\u003e \u003cb\u003eBX6\u003c/b\u003e \u003cb\u003eimpairs callose deposition\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the regulatory role of benzoxazinoids in plant defense, we evaluated callose deposition in the leaves of SV WT or \u003cem\u003ebx6-3\u003c/em\u003e plants under aphid feeding. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e, callose deposition was induced upon aphid feeding in both genotypes of the plants, with significantly lower levels observed in the mutant plants. Interestingly, the callose deposition was lower in the control leaves of \u003cem\u003ebx6-3\u003c/em\u003e plants compared to SV WT control leaves. Moreover, quantification of callose spots showed that the number of callose particles was higher in both control and aphid-treated leaves of SV WT compared to the control or aphid-treated leaves of \u003cem\u003ebx6-3\u003c/em\u003e plants, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSilencing\u003c/b\u003e \u003cb\u003eBX6\u003c/b\u003e \u003cb\u003ereduced plant tolerance to salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test whether BXDs are involved in plant response to salt stress, wheat plants (SV WT and \u003cem\u003ebx6-3\u003c/em\u003e) were irrigated with 150 mM NaCl. To assess the physiological and biochemical responses under salt stress, several parameters were measured in salt-stressed 14-day-old wheat plants. These included total chlorophyll, plant biomass, relative water content in the leaf and root, membrane stability, and reactive oxygen species (ROS), specifically hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e, all parameters measured were affected by either individual salt stress, genotype or the interaction of both these factors. Total chlorophyll content was significantly reduced in salt-stressed leaves of either WT or \u003cem\u003ebx6-3\u003c/em\u003e plants, with no significant interaction between genotype and salt treatment. Similarly, the shoot biomass was affected by salt stress or genotype, but no interaction effect was observed. Next, salt stress negatively impacted the water content in the leaf in both WT and mutant plants with no effect of genotype or interaction effect. In addition, the water content in the root was significantly lower in the salt-stressed mutant plants with a simple \u003cem\u003et\u003c/em\u003e-test, however; ANOVA showed no effect of either genotype or salt stress. While electrolyte leakage and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels increased in both WT and mutant leaves under salt stress, they were significantly affected by genotype, salt stress, and their interactions. The mutant plants exhibited a 1.1-fold higher electrolyte leakage and a 1.3-fold increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (approximately 90 \u0026micro;mol/g FW higher) compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). These results suggest that membrane integrity is more severely compromised in the mutant plants as they experienced greater oxidative stress under salt stress conditions. All in all, the \u003cem\u003eBX6-\u003c/em\u003edeficient mutant plants were more susceptible to salt stress than WT plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDue to the sessile nature of plants and the adverse effects of climate change, plants are constantly exposed to environmental biotic and abiotic stresses. Understanding the roles of specialized metabolites in plant response to abiotic and biotic stresses is crucial to comprehending their impact on (agro)ecosystem functioning and diversity. In this study, we investigate the potential role of benzoxazinoids in conferring salt tolerance and herbivore resistance in wheat.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAdverse effects of salt stress on wheat plant physiology\u003c/h2\u003e \u003cp\u003eSalt stress poses a significant challenge to durum wheat production, particularly in arid and semi-arid regions where irrigation with poor drainage leads to salt accumulation. Salt stress negatively impacts plant physiology and biochemical processes, thereby impairing cellular mechanisms and metabolism. Exposure to this stress can cause both osmotic and oxidative stress in plants. Plants exposed to salt stress, typically ranging from 50 to 200 mM of NaCl, often exhibit a decrease in chlorophyll levels, which limits photosynthetic efficiency, thereby reducing overall biomass production [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. The decrease in chlorophyll can be associated with oxidative stress caused by the accumulation of reactive oxygen species, which can lead to cell or chloroplast damage [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Moreover, salt stress critically affects water content in the leaves and roots, leading to wilting and reduced leaf expansion [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This stress also increases electrolyte leakage, indicating membrane damage, compromised cell integrity, and stress-induced injury [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. In our experiments, we also observed the negative impact of salt stress on the physiology of wheat seedlings, regardless of genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The critical effects of reduced chlorophyll content, reduced water content in leaves and roots, and increased levels of electrolyte leakage and ROS in response to the applied abiotic stress, emphasize the importance of understanding the physiological response of plants to develop breeding strategies that will improve abiotic stress tolerance [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBenzoxazinoids are involved in both herbivore resistance and salt tolerance in wheat\u003c/h2\u003e \u003cp\u003ePlant specialized metabolites are well recognized for their roles in defense against biotic stress, but they also contribute significantly to tolerance of abiotic stresses [\u003cspan additionalcitationids=\"CR75 CR76\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. These metabolites, particularly DIMBOA-Glc, are abundant in young leaves and decrease as the plant develops [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], a pattern we also observed in our study (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Accordingly, all experiments were conducted on two-week-old wheat seedlings. BXDs have previously been reported to be induced by drought, salt and aphid stress [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Our study also demonstrates that BXD levels in the leaves and roots of tetraploid wheat are modulated by cell sap sucking herbivores and salt stress, highlighting their broader involvement in wheat stress responses.\u003c/p\u003e \u003cp\u003eBXDs have been reported to exhibit antifeedant, antixenotic, antimicrobial, and allelopathic activities under biotic stresses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], but their functions in plant responses to abiotic stresses remains are still largely unexplored. In maize, DIMBOA-Glc levels are negatively correlated with susceptibility to \u003cem\u003eR. maidis\u003c/em\u003e aphids [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Aphid resistance in maize and wheat is associated with the abundance of DIMBOA and DIMBOA-Glc and the induction of callose deposition [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Callose deposition, triggered by aphid feeding, is a key phloem defense response that seals sieve pores to restrict nutrient flow to piercing-sucking arthropods [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Recently, we demonstrated that drought stress and aphid feeding enhance BXD accumulation and callose deposition in the leaves of hexaploid wheat seedlings, which correlated with reduced aphid performance [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Here, we provided evidence for the involvement of BXDs in callose deposition upon aphid feeding using a stable \u003cem\u003ebx6\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003e), thereby validating the role of BXDs in wheat defense against phloem feeding insects such as \u003cem\u003eR. padi\u003c/em\u003e aphids and potentially other cell-sap-feeding insects.\u003c/p\u003e \u003cp\u003eBXDs are also involved in plant defense against leaf-chewing insects [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. However, the effect of BXDs on caterpillars of leaf-chewing insects may be species-specific. For example, Shavit \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] reported that silencing of wheat \u003cem\u003eBX6\u003c/em\u003e in \u003cem\u003eT. aestivum\u003c/em\u003e showed no change in the body weight of caterpillars of generalist \u003cem\u003eS. littoralis\u003c/em\u003e. In contrast, another study showed better performance of the same caterpillar species on \u003cem\u003eTaMYB31\u003c/em\u003e-silenced \u003cem\u003eT. aestivum\u003c/em\u003e plants [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In another report, Li \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] showed that overexpression of \u003cem\u003eZmBX12\u003c/em\u003e in \u003cem\u003eT. aestivum\u003c/em\u003e did not affect the body weight of \u003cem\u003eS. littoralis\u003c/em\u003e caterpillars, whereas the caterpillars showed preferred feeding and more significant consumption area on wild-type plants than \u003cem\u003eBX12\u003c/em\u003e-overexpressed plants. In our study, the performance of specialist \u003cem\u003eS. frugiperda\u003c/em\u003e caterpillars was unaffected by \u003cem\u003eBX6\u003c/em\u003e-deficiency. Yet, we observed the accumulation of DIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc, HDMBOA-Glc, and HM\u003csub\u003e2\u003c/sub\u003eBOA-Glc in leaves of SV WT plants upon \u003cem\u003eS. frugiperda\u003c/em\u003e herbivory, which is in line with previous findings in maize that feeding of \u003cem\u003eS. littoralis\u003c/em\u003e and \u003cem\u003eS. frugiperda\u003c/em\u003e caterpillars on maize seedlings highly induced DIMBOA-Glc methylation and the production of HDM\u003csub\u003e2\u003c/sub\u003eBOA-Glc [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Taken together, as it was suggested by Li \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], DIMBOA-Glc \u003cem\u003eO\u003c/em\u003e-methylation increases the plant\u0026rsquo;s resistance to caterpillars through antixenosis but decreases resistance to aphids through antibiosis.\u003c/p\u003e \u003cp\u003eRecent studies have shown that the two-spotted mite (TSSM, \u003cem\u003eT. urticae\u003c/em\u003e) causes damage to cereal crops such as maize, rice, barley, and wheat, making it a potentially significant pest for these crops [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. TSSM feeding on \u003cem\u003eBXD\u003c/em\u003e-deficient mutant plants, including \u003cem\u003eBX1\u003c/em\u003e, \u003cem\u003eBX2\u003c/em\u003e, and \u003cem\u003eBX6\u003c/em\u003e Ds transposon insertion mutants in maize or VIGS-silenced \u003cem\u003ebx6\u003c/em\u003e or \u003cem\u003eTaMYB31\u003c/em\u003e wheat plants have been shown to have an increased number of progenies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. We also observed higher survival rates and egg numbers laid by adult TSSM females when fed on the \u003cem\u003ebx6-3\u003c/em\u003e mutant plants than those fed on the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that BXDs inhibit TSSM colonization. These findings highlight the potential role of BXDs in wheat defense against cell-content-feeding arthropods such as TSSM, warranting further investigation.\u003c/p\u003e \u003cp\u003eThe roles of plant specialized metabolites in abiotic stress tolerance remain largely unexplored, with only a limited number of studies investigating their involvement in plant responses to such stresses. For instance, a study showed that both flavonoid levels and the expression of key genes for flavonoid biosynthesis increased in \u003cem\u003eAtriplex canescens\u003c/em\u003e plants under salt conditions. Notably, overexpression of chalcone isomerase, a key enzyme in flavonoid biosynthesis, in Arabidopsis reduced ROS accumulation and mitigated membrane damage, suggesting that flavonoids contribute to salt tolerance in \u003cem\u003eA. canescens\u003c/em\u003e through ROS scavenging [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Similarly, a study by Batyrshina \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] reported significant accumulation of BXDs in the leaves of hexaploid wheat seedlings germinated on salt solution, indicating a possible role in salt tolerance. This response was notably stronger than that induced by polyethylene glycol (PEG) and low-temperature treatments. In line with these findings, the present study demonstrates that BXD levels are modulated in both the leaves and roots of tetraploid wheat seedlings in response to salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, \u003cem\u003eBXD-\u003c/em\u003edeficient mutant plants lacking a key biosynthetic gene (\u003cem\u003eBX6\u003c/em\u003e) exhibited greater reductions in chlorophyll, biomass, and water content in the leaves and roots, along with increased electrolyte leakage and ROS accumulation under salt stress. These results suggest that elevated BXD levels under saline conditions may contribute to oxidative stress tolerance by enhancing the plant's ROS-scavenging capacity and protecting against lipid peroxidation and other forms of oxidative damage. However, further studies are required to fully elucidate the underlying mechanisms.\u003c/p\u003e \u003cp\u003eIt is important to note that, in contrast to Shavit \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], who used VIGS to transiently suppress \u003cem\u003eBX6\u003c/em\u003e in hexaploid wheat (\u003cem\u003eT. aestivum\u003c/em\u003e), this study employed stable CRISPR/Cas9 knockout lines generated on the background of tetraploid wheat (\u003cem\u003eT. turgidum\u003c/em\u003e), allowing the assessment of a complete and heritable loss of function. Although both studies performed insect assays using the same species and observed comparable susceptibility outcomes, the use of stable mutants provides stronger genetic evidence for the role of \u003cem\u003eBX6\u003c/em\u003e in defense. Additionally, this work extends previous findings by examining the abiotic stress responses of the mutants: \u003cem\u003ebx6\u003c/em\u003e seedlings exhibited increased sensitivity to salt stress, offering functional insight not addressed in earlier studies.\u003c/p\u003e \u003cp\u003eIt is also worth noting that the accumulation of DIBOA-Glc in the \u003cem\u003ebx6\u003c/em\u003e mutant could contribute to the observed defense or stress responses. As reported by Tzin \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], the effects of BXDs on \u003cem\u003eR. maidis\u003c/em\u003e aphids and two-spotted spider mite progeny were stronger in \u003cem\u003ebx1::Ds\u003c/em\u003e and \u003cem\u003ebx2::Ds\u003c/em\u003e mutants than in \u003cem\u003ebx6::Ds\u003c/em\u003e transposon insertion mutants in maize. This pattern suggests substantial functional redundancy in the BXD pathway; notably, the partially toxic DIBOA-Glc is still produced in the \u003cem\u003ebx6\u003c/em\u003e mutant, which may contribute to residual defense activity. Therefore, while the loss of methoxylated BXDs likely represents the primary driver of the \u003cem\u003ebx6\u003c/em\u003e phenotype, a contributory role of elevated DIBOA-Glc cannot be completely excluded.\u003c/p\u003e \u003cp\u003eOverall, the combination of stable genome editing and expanded phenotypic characterization reveals new aspects of \u003cem\u003eBX6\u003c/em\u003e function in both abiotic and biotic stress responses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we provide evidence for the involvement of BXDs in both \u003cem\u003eT. turgidum\u003c/em\u003e wheat herbivore resistance and salt stress tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e). While adverse effects of salt stress were also observed in wild-type wheat, a more severe impact of the salt stress was found in the mutant plants lacking a key BXD-biosynthetic gene, \u003cem\u003eBX6\u003c/em\u003e. The \u003cem\u003ebx6-3\u003c/em\u003e plants also showed enhanced susceptibility to aphid and mite feeding. Overall, our results indicate that BXDs play an important role in wheat response to salt stress through ROS scavenging and the key enzyme gene \u003cem\u003eBX6\u003c/em\u003e in BXDs biosynthesis pathway of wheat has the potential to improve the stress resilience of cereal crops.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eBX\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBenzoxazinoid biosynthetic genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBXDs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBenzoxazinoid metabolite compounds\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRISPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClustered, regularly interspaced short palindromic repeats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDDW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDouble distilled water\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIBOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,4-dihydroxy-1,4-benzoxazin-3-one\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIBOA-Glc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIM\u003csub\u003e2\u003c/sub\u003eBOA-Glc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,4-dihydroxy-7,8-dimethoxy-1,4-benzoxazin-3-one glucoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIMBOA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDIMBOA-Glc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydrogen peroxide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHDMBOA-Glc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHM\u003csub\u003e2\u003c/sub\u003eBOA-Glc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-hydroxy-7,8-dimethoxy-2h-1,4-benzoxazin-3(4h)-one glucoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNaCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eO\u003c/em\u003e-methyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative Real-time polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eR. padi\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eRhopalosiphum padi\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eS. frugiperda\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSvevo\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eT. turgidum\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eTriticum turgidum\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTSSM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTwo-spotted spider mite\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was funded by the German Research Foundation DFG (grant no. KO 4781/4-1) and the Israel Science Foundation (grant No. 329/20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Prof. Shimon Rachmilevitch for helping us with carrying \u003cem\u003ebx6\u003c/em\u003e seeds from Prof. Distelfeld\u0026rsquo;s lab to Prof. Tzin\u0026rsquo;s lab.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe German Research Foundation DFG (grant no. KO 4781/4-1) and the Israel Science Foundation (grant No. 329/20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor information\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrench Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben Gurion, 8499000, Israel\u003c/p\u003e\n\u003cp\u003eLet Kho Hao, Reut Shavit, Beery Yaakov, Vered Tzin\u003c/p\u003e\n\u003cp\u003eLaboratory and Pilot Plant Manager, ALGIECEL Company, Copenhagen, Denmark\u003c/p\u003e\n\u003cp\u003eReut Shavit\u003c/p\u003e\n\u003cp\u003eDepartment of Entomology, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel\u003c/p\u003e\n\u003cp\u003eAdi Kliot, Murad Ghanim\u003c/p\u003e\n\u003cp\u003eDepartment of Evolutionary and Environmental Biology, and Institute of Evolution, University of Haifa, Haifa, Israel\u003c/p\u003e\n\u003cp\u003eAssaf Distelfeld\u003c/p\u003e\n\u003cp\u003eMax Planck Institute for Chemical Ecology, Department of Natural Product Biosynthesis, D-07745, Jena, Germany\u003c/p\u003e\n\u003cp\u003eTobias G. K\u0026ouml;llner\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.K.H. and V.T. conceived and designed the study. L.K.H. developed the methodology, performed the experiments, and curated the data. R.S, L.K.H, and A.D developed stable \u003cem\u003ebx6\u003c/em\u003e mutants, contributed to manuscript review and editing. L.K.H. prepared the original draft of the manuscript. V.T. managed the project, provided resources, supervised the research, manuscript review, and editing. A.K and M.G. contributed to experiments with insects, manuscript review, and editing. T.G.K. provided resources and assisted with manuscript review and editing. B.Y. contributed to reviewing and editing the manuscript. All authors read, reviewed, and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorrespondence to Dr. Beery Yaakov,\u0026nbsp;\u003c/em\u003e\u003cem\[email protected]\u003c/em\u003e\u003cem\u003e, +972544727025\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFAOSTAT. 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Plant Cell Rep. 2024;43:1\u0026ndash;17. https://doi.org/10.1007/s00299-023-03087-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Plant defense, stress resilience, benzoxazinoids, BX6, herbivore resistance; Triticum turgidum","lastPublishedDoi":"10.21203/rs.3.rs-9235518/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9235518/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBenzoxazinoids (BXDs) are a class of indole-derived specialized metabolites with primarily defensive roles against herbivores and are found in both monocots and eudicots. We recently demonstrated that BXDs contribute to drought tolerance and aphid resistance in hexaploid wheat. In this study, we investigated the potential roles of BXDs in tetraploid wheat resistance to herbivores with different feeding modes and in tolerance to salt stress using a stable \u003cem\u003eBXD-deficient\u003c/em\u003emutant. We first observed accumulation of BXDs upon insect herbivory and salt stress in wheat. To investigate BXD's function, we knocked out the key biosynthetic gene, \u003cem\u003eBX6\u003c/em\u003e, in tetraploid wheat (\u003cem\u003eTriticum turgidum\u003c/em\u003ecv. Svevo) using CRISPR-Cas9. BX6 deficiency affected herbivore performance depending on feeding mode: sucking herbivores like aphids and two-spotted spider mites performed better on mutant plants than on wild-type plants, while chewing herbivores like moth caterpillars' growth was unaffected. Under salt stress, the mutant plants showed a significant reduction in total chlorophyll, biomass, and water content in both leaves and roots compared to wild-type plants. Additionally, salt-stressed mutant plants had higher levels of electrolyte leakage and hydrogen peroxide compared to the wild type, indicating aggravated cell membrane damage and elevated oxidative stress, likely due to impaired detoxification of reactive oxygen species. These findings suggest that BX6-derived BXDs are essential for wheat herbivore resistance and salt stress tolerance. This study expands our understanding of the multifaceted roles of BXDs in stress resilience and highlights their potential for improving plant adaptation to environmental challenges and climate change.\u003c/p\u003e","manuscriptTitle":"BX6-dependent benzoxazinoid biosynthesis enhances herbivore resistance and salt stress tolerance in durum wheat Triticum turgidum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-20 09:09:46","doi":"10.21203/rs.3.rs-9235518/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"8136557997001649662479045212855505054","date":"2026-05-04T12:57:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T01:49:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154174371951849938072907500561787483467","date":"2026-04-13T02:39:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-10T10:46:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-10T09:50:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-08T11:45:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T09:34:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-04-08T08:39:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6a269412-2dae-46ac-9e81-d65f9ffa49bb","owner":[],"postedDate":"April 20th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"8136557997001649662479045212855505054","date":"2026-05-04T12:57:56+00:00","index":58,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T01:49:09+00:00","index":36,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T09:09:52+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-20 09:09:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9235518","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9235518","identity":"rs-9235518","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}