20 µM SL application very significantly (****p < 0.0001) increased gene expression, particularly for LsABCC3, LsCCD7, LsCCD8, LsD27, LsMAX1, and LsMAX2 in shoot and root tissues compared to the control (Figs. 5 and 6). The increase in LsABCC3, LsD27, and LsMAX1 gene expression was especially notable, reaching about 21-fold in the shoot under 20 µM SL (Figs. 5 and 6). Only Pb stress significantly reduced the expression of most genes, particularly LsCCD8, and LsD14 in a tissue-dependent manner (Figs. 5 and 6). The only exception was the LsMAX2 gene, which showed twofold higher expression in both root and shoot under stressful conditions (Fig. 6). Additionally, the expression levels of LsHMA2 and LsMAPK genes were upregulated in only shoot tissues under stress conditions (Fig. 6). Interestingly, the combined Pb and SL treatment (Pb + 20 µM SL) very significantly (****p < 0.0001) increased the expression of LsMAX2 in both tissues by about threefold (Fig. 6).
Increasing heavy metal pollution in agricultural soil causes toxicological effects on soil-plant systems and humans. Among all heavy metals, Pb is known as a persistent heavy metal that has significant toxicological effects on ecosystems. The mobility of Pb from soil to the plant system is a major concern for its bioaccumulation in plants and its stability in the food chain, which may eventually disrupt related biosystems. Using phytohormones is a good way to help plants cope with heavy metal (HM) stress, providing a sustainable method that has little effect on the environment. Strigolactones (SLs) are a recently identified phytohormone that has been found to have potential application in plant growth and development. In the study, the potential role of SLs in mitigating the detrimental effects of Pb toxicity on lettuce was comprehensively evaluated.
Pb is not an essential plant nutrient and therefore high Pb content negatively affects the morphological, physiological and biochemical properties of plants, reducing product production and yield. Elevated concentrations of Pb in the environment have an adverse impact on key plant metabolic activities, including germination, growth, photosynthesis, nutrient uptake, and overall biomass accumulation. Moreover, Pb toxicity delays root formation through increased carbohydrate and protein content by affecting polyphenol oxidases and peroxidase activity. It was found that Pb stress significantly negatively affected plant development (fresh and dry biomass, leaf area, root properties) in lettuce (Tables 3 and 4). Moreover, Pb content has shown a significant negative correlation with biomass and root morphology characters. Similarly, previous studies have reported that Pb stress negatively affects the development of lettuce and some other plants. It was determined that SL applications reduced the negative effects of lettuce seedlings on plant growth parameters (Tables 3 and 4). SLs have been suggested to restore tomato plant growth by alleviating the phytotoxic effects of Cd on photosynthetic pigments and gas exchange parameters. The exogenous application of SLs may be a promising strategy to reduce Cr uptake and alleviate its harmful impacts on tomato plants cultivated in polluted soils. Likewise, various studies have demonstrated that strigolactones (SLs) can alleviate stress and enhance plant growth in species such as grape, tomato, soybean and cucumber, under different stress conditions by boosting photosynthetic efficiency, protecting the photosynthetic apparatus, stimulating root hair and lateral root formation, and enhancing antioxidant defenses. SLs are thought to play a role in regulating root system architecture, especially in response to nutrient availability and environmental stresses.
Relative water content (RWC) is widely accepted as a reliable indicator of plant water status, as it effectively represents the equilibrium between water uptake and loss via transpiration. Relative to control plants, Pb stress resulted in a substantial reduction (- 18.73%) in LRWC. Additionally, when plants were under Pb stress, LRWC exhibited a strong response to 10 μL and 20 μL SL applications, resulting in increases of + 10.6% and + 11.39%, respectively, compared to the stressed control (Table 5). Various doses of Pb(NO₃)₂ treatments resulted in a reduction in RWC and chlorophyll pigment levels in broad bean plants. SL applications have been shown to reduce the negative effects of Cd stress in radish, with a 6% increase in RWC in plants treated with 25 µM exogenous SL compared to those that were not treated under Cd stress. Toxic metal exposure adversely affects photosynthesis by decreasing the contents of chlorophyll and carotenoids, primarily through the inhibition of enzymes involved in their biosynthetic pathways. Pb can impair photosynthetic efficiency by diminishing the activity of key enzymes involved in the Calvin cycle, such as ribulose-1,5-bisphosphate carboxylase (Rubisco). The presence of Pb has been reported to inhibit key enzymes responsible for photosynthetic CO₂ assimilation, such as Rubisco and ribulose-1,5-bisphosphate kinase. In lettuce seedlings grown under Pb stress, Clo a, Clo b and total chlorophyll contents decreased, but with SL application, Clo a, Clo b and total chlorophyll contents increased (Table 5). Mallhi et al. reported that Pb stress decreased the photosynthetic properties (chlorophyll and gas exchange parameters), along with a decrease in biomass and growth in Ricinus communis L. The application of SLs can increase photosynthesis by promoting the development of chloroplasts, which results in an increase in chlorophyll content. The authors suggested that the increase in pigments under stress with SL applications might be due to the decrease in SL-induced oxidative damage.
Stressful environmental conditions trigger excessive ROS generation, which impairs photosynthetic efficiency, hampers plant growth, and elevates oxidative damage at the cellular membrane level. This investigation demonstrated a substantial increase in ROS, as seen by elevated levels of EC, H₂O₂, and MDA (Tables 5 and 6). Disruption of ROS homeostatic balance may have resulted in increased EL, HO and MDA levels in leaves. Sharma et al. observed that the significant elevation in EL levels following Pb application underscores the disruption of membrane integrity and highlights the oxidative stress induced by Pb in barley plants under Pb stress conditions. Mallhi et al. reported that different levels of Pb (0, 300, 600 mg/kg soil) applications in Ricinus communis L. induced oxidative stress in plants, including HO and MDA production. A previous study revealed that metal stress led to an elevation in ROS, as evidenced by increased electrolyte leakage, MDA, and H₂O₂ levels. Plants generally face oxidative damage when exposed to Pb and other metals. In this study, Pb accumulation correlated with stress-related characters like H₂O and MDA, showing a strong positive correlation. Moreover, these characters were located close to the Pb application in PC1. Recent studies indicate that when plants face salty conditions, SLs activate a process that includes nitric oxide (NO), ROS, and Ca⁺ signaling, all of which work together to reduce damage from oxidation in plant cells. Ca⁺ ions function as ubiquitous secondary messengers in plant responses to abiotic stress, and their presence has been shown to effectively suppress the accumulation of sodium ions (Na⁺), hydrogen peroxide H₂O₂, and MDA, thereby contributing to enhanced stress tolerance mechanisms. The exogenous application of SL effectively alleviated oxidative stress in lettuce plants under Pb stress by significantly reducing EC, H₂O₂, and MDA levels, suggesting its potential role in mitigating Pb-induced cellular damage (Table 6). When maize plants were exposed to 100 mM NaCl, the levels of H₂O₂ and MDA increased a lot, showing more oxidative stress; however, treating them afterward with 0.01 mg/L GR24 (a synthetic SL analogue) greatly lowered the amounts of H₂O₂ and MDA in two types of maize.
Plants respond to environmental stresses by synthesizing a variety of metabolites, including proline, polyamines, asparagine, serine, sugars such as glucose and fructose, as well as antioxidant molecules that regulate ROS levels. Proline and soluble sugars are well-known to accumulate in plant cells under various environmental stress conditions, including drought, salinity and heavy metal and they play crucial roles in enhancing stress tolerance. These osmolytes contribute significantly to osmotic balance, enzyme and protein stabilization, ROS detoxification, regulation of cellular redox homeostasis, protection of thylakoid membranes, and preservation of photosynthetic performance. Under Pb stress, proline accumulation is regulated through the activation of key enzymes such as ornithine-δ-aminotransferase and γ-glutamyl kinase, which are involved in the biosynthetic pathways of ornithine and glutamate. In the study, Pb stress increased the proline content, and SL treatments decreased the proline content under Pb stress compared to Pb treatment alone. In addition, Pb stress increased sugar content compared to the control, but SL treatments under Pb stress caused a further increase in sugar content (Table 6). The increased levels of soluble sugars and proline, along with enhanced leaf relative water content, indicate that GR24 may alleviate abiotic stress by promoting osmotic regulation.
Plants develop ROS scavenging systems to protect cell membranes from oxidative stress, utilizing antioxidant substances and enzymes like SOD, CAT, POD, APX, GR and GST. SOD plays a crucial role in ROS scavenging by controlling O₂⁻ levels produced in the mitochondria, chloroplasts, and cytosol. CAT is involved in eliminating photorespiratory H₂O₂, contributing to cellular ROS detoxification. POD contributes to HO removal by oxidizing diverse substrates, producing water and corresponding oxidized compounds. APX exhibits a higher affinity for H₂O₂ than CAT and reduces H₂O₂ to water by using ascorbate as an electron donor across various subcellular compartments. The results showed that Pb stress impacts all enzyme activities, either alone or under SL application. Pb stress led to higher levels of CAT, POD, SOD, APX, GR, and GST in lettuce, (Tables 7 and 8). In the study conducted on cotton, it was determined that heavy metal stress increased antioxidant enzymes such as SOD, POD, CAT and APX in roots and leaves. The Pb treatments increased the antioxidant enzyme activities, such as SOD, POD, and CAT, in rice (Khan et al.). Although SL treatments decreased their activity under Pb stress, they still showed higher activity than the control treatment (without Pb stress). Both doses of SL treatment together with Pb treatment decreased SOD, CAT, POD and APX enzyme activities and only Pb + 10 µM SL treatment reduced the activity of GR enzyme. This might happen because ROS is removed without enzymes by boosting the effects of non-enzymatic antioxidants or by increasing the activity of other antioxidant enzymes. Plants synthesize soluble antioxidants such as glutathione (GSH) and ascorbate (AsA) to enhance their antioxidant defense system, allowing them to cope with environmental stress. GR is an essential enzyme that reduces oxidized glutathione (GSSG) back to its active, reduced form (GSH) as part of the AsA-GSH cycle, thereby maintaining cellular redox homeostasis. The GST enzyme plays a crucial role in detoxifying ROSs by catalyzing the conjugation of reduced GSH to various electrophilic compounds. SL treatments (especially Pb + 20 µM SL) significantly increased the activity of GR and GST, in lettuce seedlings under Pb stress (Table 7). Similarly, when exposed to cadmium stress, Vigna angularis exhibits a notable increase in the activity of AsA-GSH cycle enzymes, demonstrating its ability to cope. SL enhances stress tolerance by maintaining osmotic balance, limiting Cr translocation, enhancing antioxidant enzymatic activity, and redox balance, thus protecting plant cells from oxidative damage Under Cr stress, AsA-GSH with SL treatment generates higher GSH levels due to biosynthesis and regulation of GR activity, potentially facilitating efficient hydroperoxide scavenging by GST and GPX. SLs have also been reported to increase antioxidant-related gene expression in Brassica napus, Vitis vinifera, and rice.
The oxidative pentose phosphate pathway (OPPP) is a ubiquitous and crucial metabolic route in higher plants, including species such as barley, soybean, Arabidopsis, and tobacco, where it plays a vital role in maintaining cellular redox balance under stress conditions. Pb treatment increased the activities of G6PD and 6GPD enzymes and decreased the activity of the NRA enzyme (Table 8). G6PDH is a key enzyme in the OPPP, and it controls the amount of nicotinamide adenine dinucleotide phosphate (NADPH) by adjusting how glucose is processed in OPPP. 6GPD is a key enzyme in the OPPP that catalyzes the third step of the pathway. 6PGDH reduces NADP⁺ to NADPH while converting the 6-phosphogluconate molecule to ribulose-5-phosphate. Previous studies have demonstrated that G6PDH is involved in responses to various environmental stresses, including UV-B radiation, heavy metals, salt, heat and drought stress. Tian et al. reported that under cold stress, the expression levels of TaG6PDH and Ta6PGDH, two key enzyme genes of the OPPP, were significantly upregulated in the tillering nodes and leaves of Dn1. The increase in G6PDH and 6PGDH activities in hydroponically grown barley plants after cold stress (4 °C, 1 day) revealed that the oxidative phase of OPPP plays an essential role in the cold stress response. Nitrate reductase (NR) is a substrate-induced enzyme that catalyzes the reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻), thereby facilitating the production of NO. High chloride (Cl⁻) stress significantly inhibited nitrate reductase activity (NRA) in maize, likely due to the antagonistic interaction between Cl⁻ and NO₃⁻ uptake. Even though Pb + SL treatments raised G6PD activity more than the control group, the biggest increase compared to the Pb control was seen in the 6GPD and NRA enzymes (Table 8). Nickel (Ni) toxicity markedly reduced NRA in both Dunkeld and Shiralee cultivars, while taurine treatment significantly alleviated this inhibition by enhancing NRA levels under Ni stress. Exogenous SL causes NO synthesis, implying negative feedback control of SL levels. NO acts as an upstream regulator of SL signaling, with the nature of their interaction being influenced by nutrient availability.
Our findings indicate that lettuce plants under Pb stress showed a significant decrease in leaf N, P, K, Ca, Mg, Fe, Zn, and Cu contents as well as an increase in Pb accumulation (Table 9). Moreover, elevated Pb concentrations in the soil reduce the availability of other essential nutrients (Ca, Fe, Mg, Mn, P, and Zn) by either obstructing their uptake or binding them to ion carriers. Lamhamdi et al. reported that high Pb concentration causes a decrease in most macro elements (K, Ca, P, Mg and Na) and micro elements (Fe, Cu and Zn). Therefore, the suppression of mineral ion uptake seems to be a common outcome of Pb exposure. In contrast, the administration of 20 μL SL significantly improved nutrient acquisition and decreased Pb uptake and accumulation in plants grown under Pb toxicity (Table 9). SLs facilitate the response of plants to N and P starvation by shaping the aboveground and belowground architecture. SLs affect various characteristics of root development to manage the architecture of the general root system. In legumes, more nodules help them take in nutrients better, while in non-legumes, a lack of P and N causes them to release SL to encourage arbuscular mycorrhizal (AM) partnerships for getting nutrients. The enhanced nutrient uptake observed with SL applications may be attributed to the hormone's influence on root architecture, leading to improved root growth and development. GR24 application markedly enhances callus biomass, boosts antioxidant enzyme activities, elevates stress-associated metabolite levels, and promotes the accumulation of K⁺ and Ca⁺ ions in sunflower. In switchgrass seedlings that were exposed to Cd, the absorption of Zn, Fe, and Cu in both the roots and above-ground parts was greatly lowered; however, adding GR24 significantly improved the uptake of these micronutrients in both areas.
Cytosolic HM ions, like Pb, can be kept out of the apoplasts by ATP-binding cascade (ABC) transporters found at the plasma membrane, which are essential for moving HMs, including Pb, from the roots to the above-ground parts of plants. Many ABC transporters have two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs), making them full-size, whereas half-size transporters only have one NBD or TMD. Arabidopsis ATP-binding cassette C subfamily 1, 2 and 3 (ABCC1, ABCC2, and ABCC3) are transporters that facilitate the movement of glutathione conjugates. Additionally, ABCC1 is involved in transporting folate and metals, while ABCC2 and ABCC3 are engaged in transporting chlorophyll breakdown products. Although Cd treatment did not affect the expression of abcc1 and abcc2 genes in A. thaliana, it led to an increase in abcc3 transcript levels. Contrary to these findings, our study showed that Pb treatment did not affect ABCC3 gene expression in lettuce compared to the control (Fig. 5). Experimental data show that Pb treatment has a very mild effect on the ABCC3 gene regulatory pathway. The study's results revealed that only SL treatments increased the expression of the ABCC3 gene in both tissues compared to the control (Fig. 5). SL applications may increase the expression of the ABCC3 gene by activating defense and detoxification pathways in the plant. ABCC3 acts as a detoxifier by helping to store glutathione compounds and heavy metal complexes in vacuoles.
SLs are mainly synthesized in roots via the plastid-localized conversion of all-trans-β-carotene to carlactone by the sequential action of CCD7, CCD8, and D27 enzymes, and then transported to aerial parts. CCD7 and CCD8 expressions are modulated by nutritional status, light, and other factors to help plants adjust their architecture under unfavorable conditions. It was observed that the LsCCD7 gene exhibited an increased expression profile, especially in root tissue, in parallel with increasing SL application concentrations, both alone and in combination with Pb application (Fig. 5). Again, according to the expression analysis result of the LsCCD7 gene, it was determined that Pb application had the lowest expression profile in the shoot tissue (Fig. 5). In addition, LsCCD8 gene had the highest expression profile in both tissue types and in 20 μM SL applications. This increase was found to be greater in the root tissue than in the shoot tissue (Fig. 5). It is thought that the probable reason for this increase in the root is that the SL hormone is first synthesized in the roots and then transported to the above-ground parts of the plant. In rice, OsCCD7 and OsCCD8 transcripts are predominantly expressed in the vascular parenchyma cells of roots. These increases in our findings were recorded to be very significant (****p < 0.0001) according to statistical analysis. On the other hand, it was found that the LsCCD8 gene had the lowest expression profile in both tissue types in Pb application. However, it was noted that SL applications together with Pb application increased the expression profile of the LsCDD8 gene in both shoot and root tissue compared to Pb application (Fig. 5). Many studies in plants have reported that CCD8 genes exhibit an increasing or decreasing expression profile according to the stress group and severity. In the grapevine, drought stress led to a tissue-specific response where VvCCD7 and VvCCD8 transcript levels were elevated in leaves but suppressed in roots. Consistently, this response was accompanied by a notable increase in SL levels in leaves, along with enhanced expression of SL biosynthetic genes, including VvCCD7 and VvCCD8. Our study showed that when stress was applied to the roots, the expression of LsCCD7 and LsCCD8 increased significantly in the root tissues (Fig. 5) especially after applying SL, indicating that the activation of these genes is closely linked to the part of the plant that senses stress.
SL signaling is initiated by the binding of SL molecules to the α/β-hydrolase receptor DWARF14/ DECREASED APICAL DOMINANCE 2 (D14), which undergoes conformational changes that enable its interaction with other signaling components and trigger the degradation of repressor proteins. D14, with its conserved Ser-His-Asp catalytic triad, and the F-box protein MAX2 function as core components of the SL perception complex in higher plants, essential for SL hydrolysis and signaling. In rice, SL signaling involves D14, MAX2, the SCF complex (D3), and ClpATPase (D53), which regulate gene expression by degrading transcriptional repressors. There are studies on the change in expression of the D14 gene in response to various stresses, including drought, heavy metals, and light. In the study, Pb treatment significantly (****p < 0.0001) decreased LsD14 gene expression in both root and shoot tissue compared to the control. SL treatments in combination with Pb stress caused a significant increase in the LsD14 gene, especially in shoot tissue, compared to Pb stress (Fig. 5). The activation of D14 is initiated by the intact SL molecule, which induces a destabilized conformation in D14 by interfering with the formation of its catalytic triad, rather than by its hydrolysis intermediates or products. When the expression profiles of the LsD27 gene in shoot and root tissues were compared to the control, it was determined that the highest increase in both tissue types occurred with the 20 μM SL application. This increase was recorded to be statistically very significant (****p < 0.0001). D27 is described as an iron-containing enzyme in the chloroplast that participates in the isomerization of carotenoids, including the conversion of all-trans-β-carotene to 9-cis-β-carotene, which is essential for subsequent CCD7 and CCD8 mediated reactions in SL biosynthesis. More recent studies have further confirmed this enzymatic role, characterizing D27 as a plastid-localized β-carotene isomerase essential for SL production. Waters et al. reported that exogenous SL application in Arabidopsis increased the expression level of the D27 gene. In parallel with this study, our findings determined that the LsD27 gene increased with the increase in SL concentration (Fig. 6). In rice, P and N deficiencies promote SL accumulation in the roots, triggering nutrient stress signaling through the upregulation of D10, D17, and D27 while downregulating the expression of D3, D14, and D53. In our study, LsD27 expression showed no significant change under Pb treatment (Fig. 6).
P1B-type heavy metal ATPases (HMAs) are membrane-bound proteins involved in maintaining metal ion balance within the cell. The N- and C-terminal regions of heavy metal-binding domains in typical P1B-type ATPases can associate to bind metal ions specifically like Cd⁺ and Pb⁺. In Pb treatment, the LsHMA2 gene increased at the highest level in both shoot and root tissues; this increase was found to be statistically very significant (****p < 0.0001) in shoot tissue and insignificant (ns) in root tissue (Fig. 6). It is thought that the reason for this increase is that the heavy metal ATPase gene family known as HMAs are heavy metal transporters that show a strong affinity to absorb and pump heavy metals into plant cells. Indeed, as a result of studies conducted in many plant species, it has been reported that heavy metal applications increase the expression profile of this gene group. In wheat, treating with 5 and 10 µM selenium reduced the activity of the TaNramp5 and TaHMA2 genes that help take in and move Cd, while it boosted the activity of the TaHMA3 gene, which moves Cd to the vacuole, in both the roots and shoots. Moreover, Sayyadi et al. reported that varying Pb concentrations increased the expression of HMA2, HMA3, and HMA4 genes in the roots and leaves of rice seedlings, with the highest increase observed at high Pb levels. In addition, sodium nitroprusside (SNP) treatment decreased HMA2 expression in roots and did not cause any significant change in leaves. In parallel with this study, we found that LsHMA2 gene expression decreased in Pb-containing SL treatments compared to Pb alone (Fig. 6).
Heavy metal stress triggers certain signaling molecules ROS and phytohormones that turn on specific transcription factors, and adding phosphate groups to proteins through mitogen-activated protein kinases (MAPKs) is an important way to control the expression of antioxidant enzymes. MAPK signaling, as one of the most prevalent pathways in plants, plays a crucial role in mediating tolerance mechanisms by functioning as a key component of signal transduction under abiotic and biotic stress conditions. Moreover, under stress conditions, MAPKs in plants can modulate various transcription factors such as bZIP, MYB, MYC, and WRKY. Increased LsMAPK gene expression was observed with Pb treatment (Fig. 6). Huang and Huang suggested that Ca⁺ ions play a role in Pb-induced cell death and trigger the MAPK pathway via CDPK by increasing the activity of CDPK-like kinases. The SlMAPK3 gene in tomatoes showed a significant increase in expression when exposed to Cd treatment. The MAPK cascade involves three parts: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK; they are linked by a phosphorylation process that sends signals to specific transcription factors (TxF). Transcriptome analysis of Raphanus sativus roots under Pb stress revealed that four MAPK genes (MAPKKK7, MAPK6, MAPK18, and MAPK20) were upregulated in response to the treatment (Wang et al.). Navarro et al. reported that MAPKs become activated upon exposure to As (III), indicating their potential involvement in arsenic-induced signaling pathways. When SL was applied together with Pb, there was a decrease in LsMAPK expression compared to this increase (Fig. 6). GR24 boosts antioxidant enzyme activity under low-temperature stress (4 °C). At the same time, RNA-seq analysis revealed altered expressions of ROS and MAPK related genes, upregulating PXG3, ISCA, ISU1, NFXL1, VTC2, and NTRC, and downregulating SF3B1, ESM1, and PAB2.
In Arabidopsis, the P450-type MAX1 enzyme converts carlactone to the intermediate methylated carlactonic acid, which binds to the SL receptor α/β-hydrolase D14. MAX genes associated with SL biosynthesis are more sensitive to abiotic stresses than those involved in SL signaling. In 20 μM SL treatment, LsMAX1 gene showed the highest expression level in both shoot and root tissue (Fig. 6). In Pb treatment, it was determined that LsMAX1 expression increased, especially in shoot tissue, compared to the control (Fig. 6). MAX1, an essential enzyme for making SL, showed increased levels when plants were under heat stress, decreased levels in plants with more AtMYBS1, and higher levels in atmybs1 mutants. On the other hand, it was determined that SL applications together with Pb applications increased the expression profile of the LsMAX1 gene (Fig. 6). Ünlü et al. reported that applying the exogenous SL analog GR24 increased the expression of the MAX1 gene in sugar beet. Castillo et al. reported that NO treatment led to a stronger upregulation of MAX1 and MAX2 gene expression in Arabidopsis seedlings. Elevated intracellular glucose levels have been shown to induce stress-related gene expression, suggesting a potential mechanistic interaction between sugar signaling and SL pathways, particularly evident in Hxk1 and Max2 mutants. In our study, treatments with Pb and Pb + SL significantly boosted sugar levels compared to the control, indicating a possible link between SL and sugar signaling when under heavy metal stress (Table 6). Supporting this, Pb + SL co-application significantly enhanced the expression of the LsMAX2 gene (Fig. 6), a key regulator of SL signaling. Consistently, MAX2 has been reported to be induced under various abiotic stress conditions, and overexpression of CsMAX2 in transgenic lines resulted in reduced MDA accumulation under salt, drought, and ABA stress, highlighting its protective role in stress mitigation. Moreover, Ma et al. demonstrated that GR24 application upregulated the expression of several SL biosynthetic genes (PbD27, PbDAD1, PbCYP711) and signaling components (PbD14, PbMAX2A, PbPDR1), further supporting the involvement of MAX2 in SL mediated stress responses.