Phytoremediation Potential and Differential Chromium (Cr) Accumulation and Gene Expression in Brassica juncea Varieties for Contaminated Soils in Turkey

Phytoremediation Potential and Differential Chromium (Cr) Accumulation and Gene Expression in Brassica juncea Varieties for Contaminated Soils in Turkey | 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 Phytoremediation Potential and Differential Chromium (Cr) Accumulation and Gene Expression in Brassica juncea Varieties for Contaminated Soils in Turkey Nuriye Merakli, Abdulrezzak Memon, Huseyin Altundag, Baris Kaki, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7303811/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Several agricultural areas in the Anatolian Plateau of Turkey are contaminated with chromium due to mining and leather tanning activities, posing a serious threat to both safe farming and human health. Cleaning polluted soils for reuse in agriculture is a vital and primary goal of this research. This study aimed to identify suitable Brassica juncea varieties for phytoremediation of metal-contaminated soils in Turkey, with the goal of promoting safe farming practices. Six varieties—Early Raya, Sindh Raya, S-9, Ganj Sarhen, JS-13, and THB-8—developed by the Agricultural Institute of Pakistan for drought and salt tolerance, were tested for metal tolerance at different Cr levels (0–1000 µM). Physiological parameters were recorded, and the contents of Cr, Ca, Fe, Mg, and K in plant tissues were quantified using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Additionally, the gene expression of several enzymes induced by ROS was examined using RT-qPCR. Early raya and Sindh raya under Cr stress showed different expression patterns of these genes, highlighting significant genotypic differences in chromium uptake, nutrient balance, and molecular responses. Our results classified the varieties into three different groups: Early raya and THB-81 as accumulators, Sindh raya, and S-9 as excluders, and Ganj sarhen, and JS13, as intermediate Cr accumulators. This study offers important insights into genotype-specific detoxification strategies. It lays the fundamental groundwork for future breeding programs and phytoremediation research aimed not only at reducing Cr contamination and promoting safe agriculture in contaminated soils in Turkey and other countries facing similar pollution issues. Brassica juncea chromium accumulation gene expression phytoremediation sustainable agriculture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In recent years, there has been significant development activity involving industrialization and rapid urbanization across nearly all of Turkey. The leather and textile industries are among the sectors that have experienced considerable growth. In many parts of Turkey, the environment has reached or exceeded its carrying capacity regarding soil and water pollutants such as lime (Ca(OH)₂), sodium sulfide (Na₂S), sodium chloride (NaCl), various dyes, and toxic heavy metals, especially chromium (Cr), released by these industrial sectors (Harmancioglu et al. 2008 ). Chromium (Cr) is one of the toxic heavy metals that can harm soil, groundwater, and ecosystems due to its widespread and unconscious use in industrial processes (Ertani et al. 2017 ; Adnan et al. 2024 ). In Turkey, several regions, especially Anatolia, have significant mining and leather tanning activities, most of which generate waste effluent that is a major source of soil and river water pollution (Akcay et al. 2003 ; Dundar et al. 2012 ; Tokatli et al. 2021 ). These activities negatively impact agriculture, animal husbandry, and the quality of drinking water by contaminating soil and rivers. Turkey's most polluted rivers include the Euphrates, Tigris, Gediz, and Menderes; although they are vital for irrigation, they pose serious risks to public health and food safety (Aydin and Kucuksezgin 2012 ; Şentürk and Yıldız 2015 ). The increasing industrial activity continues to elevate environmental pollution, with long-lasting effects on regional ecosystems (Saxena 2025 ). As industrial use of chromium grows, its presence as an environmental contaminant has increased significantly (Coetzee et al. 2020 ). Chromium lacks any essential function in plant metabolism; however, it exerts widespread phytotoxic effects that impair nearly all biological functions in plants (Shahid et al. 2017 ). Cr can interfere with plants' ability to absorb and maintain the balance of vital mineral elements. Competition with other elements interferes with physiological and metabolic functions, leading to growth suppression, oxidative damage, chlorosis, root impairment, and nutritional imbalance, which can ultimately result in plant mortality (Anjum et al. 2016 ; Singh and Malaviya 2019 ). One of the most crucial consequences of Cr stress is the impairment of antioxidant defense systems and the disturbance of the equilibrium between the generation of reactive oxygen species (ROS), which cause cellular damage (Samantary 2002 ; Saud et al. 2022 ). Chromium contamination poses a serious threat to global food security by impairing the growth and yield of essential crops, including vegetables, cereals, and legumes (Vasilachi et al. 2023 ). To reduce its harmful effects, it is crucial to investigate how chromium behaves in soil environments, assess its toxic impact on plant growth and metabolism, and develop effective, long-term remediation strategies. Among the benefits these activities can provide are improving soil quality and encouraging environmental sustainability (Xia et al. 2019 ). Recent research has focused on how crop plants absorb and accumulate chromium and how this process may affect human health through the food chain (del Real et al. 2013 ; Zulfiqar et al. 2023 ). Phytoremediation, the use of plants to extract, sequester, or detoxify pollutants, stands out as a sustainable and efficient method to clean up environments contaminated with heavy metals (Memon 2016 ; Doğru et al. 2021 ). Enhancing the phytoremediation capacity of plants requires a thorough understanding of the physiological and molecular processes that control the uptake of metals, their interaction with essential nutrients, and the mechanisms involved in detoxification (Memon and Schröder 2009 ). Additionally, identifying and utilizing promising plant genotypes that can either accumulate or exclude the target metal is crucial for successful phytoremediation. Grown for both agricultural and industrial purposes worldwide, Brassica juncea L. (Brown mustard), is an important edible oilseed crop with significant nutritional and economic value. It is also recognized for its strong ability and significant potential in phytoremediation, especially in areas affected by environmental pollution such as heavy metal-contaminated soils (Anjum et al. 2012 ). Due to its reported tolerance to several heavy metals, B. juncea is considered a sustainable and environmentally friendly option for soil remediation (Singh et al. 2024 ). Finding and selecting genotypes with natural tolerance to heavy metals is an effective way to develop improved varieties suitable for growing in areas contaminated with heavy metals (del Real et al. 2013 ). However, a notable gap remains in understanding B. juncea genotypes that can be adapted to Turkey’s specific climate conditions and provide efficient Cr control under field-related stress levels. This study was conducted to investigate the capacity of six Brassica juncea varieties, Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81, to accumulate and tolerate chromium (Cr) at different concentrations (0, 10, 50, 100, 200, 500, and 1000 µM). The study also explores the potential roles of key elements like Ca, Fe, K, and Mg in reducing Cr toxicity. To understand the molecular mechanisms that boost antioxidant defenses under chromium stress, the transcription levels of ROS-related genes—ascorbate peroxidase (APX), respiratory burst oxidase homolog (RBOH), and iron superoxide dismutase (FeSOD)—were assessed via RT-qPCR in the root tissues of the Cr-accumulator variety (Early raya) and the Cr-excluder variety (Sindh raya). Our work offers an important and practical, comprehensive, two-pronged approach for addressing contaminated sites polluted with Cr. One approach utilizes accumulator varieties for active soil remediation, whereas the other employs excluder varieties such as Sindh raya for sustainable and safe farming. This innovative, multi-varietal comparison of chromium phytoremediation provides a more thorough and practical approach to managing soil contamination, addressing cleanup, food safety, and developing effective and safe strategies for metal-contaminated soils. MATERIALS AND METHODS Plant material and experimental design Seeds of Brassica juncea (JS-13, Early raya, Sindh raya, S-9, Ganj sarhen, and THB-81) were obtained from the Agricultural Research Institute, Tandojam, Pakistan. Seeds were surface-sterilized with 5% sodium hypochlorite and Tween-20 for 15 min, then rinsed with distilled water. The plant growth conditions and cultivation methods are the same as described previously (Meraklı et al. 2022). Seeds (100 per pot) were sown in vermiculite and germinated under controlled conditions (25 ± 2°C day/20 ± 2°C night, 16 h light/8 h dark, 150 μmol/m²/s light intensity, 60 ± 5% humidity). On day 5, germinated seeds (radicle ≥ 2 mm) were counted, and the germination rate (%) was calculated according to (Akinci and Akinci 2010). Seedlings were treated with CrCl₃·6H₂O at 0, 10, 50, 100, 200, 500, and 1000 μM in 1/4-strength Hoagland solution (Hoagland and Arnon 1938). Control plants received only the nutrient solution. After two weeks, seedlings were harvested, and roots were rinsed with sterile deionized water. The experiment was performed in triplicate. Growth parameters Plants were harvested after 15 days (two weeks), carefully removed from the pots, washed to remove vermiculite residue, and separated into leaves, stems, and roots. Root and plant lengths were measured, and the fresh weight (FW) of roots and shoots was recorded after harvesting. Plant and root lengths were measured using a meter scale. The samples were oven-dried at 72°C for approximately 48 hours. Metal analysis Dried plant samples (0.5 g) were digested in a mixture of nitric and perchloric acid (HNO₃: HClO₄, 5:2 v/v) on a hot plate, then filtered through Whatman No. 42 (0.25 µm) and diluted to 10 ml with Milli-Q water (Memon et al. 1979). Metal concentrations were determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES; SPECTRO ARCOS) (Meraklı et al. 2022). Chromium standards (Sigma-Aldrich, Germany) and blank samples were prepared according to the method described by (Meraklı et al. 2022). Gene expression analysis Total RNA was extracted from the roots of control and Cr-treated plants using the RNeasy Plant Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. To eliminate genomic DNA contamination, the RNA samples were treated with RNase-Free DNase. RNA concentrations were measured using a NanoDrop spectrophotometer. cDNA synthesis was performed using the Bio-Rad iScript cDNA Synthesis Kit. The reaction mixture contained iScript reaction mix, reverse transcriptase, the RNA template, and nuclease-free water. Quantitative real-time PCR (qRT-PCR) was performed using the LightCycler 480 system (Roche, Germany) with SYBR Green Master Mix. The reaction included gene-specific primers for APX, FeSOD, RBOH, and ubiquitin. The qRT-PCR procedure followed standard protocols (Meraklı and Memon 2023). Gene-specific primers (see Table S1) were designed based on mRNA sequences obtained from the GenBank and EMBL databases. The ubiquitin gene served as the reference gene. Gene expression quantification was performed using the ΔCt method, where ΔCt values are inversely proportional to the initial template amount (Applied Biosystems). Relative gene expression levels were calculated using the 2^−ΔΔCt method. Statistical analysis Data (mean ± SD) were analyzed using SPSS (24.0) and Microsoft Excel. Tukey's test was used to evaluate differences in plant growth, and simple correlation coefficients were used to assess the significance of metal concentrations. Data normality was assessed using the Shapiro-Wilk test, and variance homogeneity was examined using Levene's test. A three-way ANOVA was used to investigate the effects of the factors and their interactions. Post hoc comparisons were performed using Duncan's test. Principal Component Analysis (PCA) was performed to identify patterns of Cr accumulation and better understand the relationships among the studied variables. The principal components that explained most of the total variance were extracted, and the distribution of samples was assessed based on Cr accumulation levels. The positioning of the different factors was visualized using biplot graphs, and the loadings of the principal components were interpreted to elucidate their contribution to the observed variation. R (version 4.2.2), Python (version 3.10.0), and SPSS (version 24.0) were used for the analyses, and p < 0.05 was considered significant. RESULTS Effects of Chromium (Cr) on growth in Brassica juncea varieties Bar charts in Fig. 1 illustrate how Cr influences various growth metrics across six Brassica juncea varieties: Early raya, Ganj sarhen, JS-13, Sindh raya, S-9, and THB-81 grown at different chromium concentrations (0, 10, 50, 100, 500, and 1000 µM). These metrics include seed germination, plant height, root length, root fresh weight, root dry weight, shoot fresh weight, and shoot dry weight (Fig. S1; Fig. 1). Across all varieties and parameters, a clear dose-dependent response to increasing chromium levels is evident. Low Cr concentrations (10, 50, 100 µM) generally have a stimulating or neutral effect on plant growth compared to the control (0 µM). However, as Cr levels rise to 200, 500, and especially 1000 µM, all growth measurements significantly decrease across all varieties, indicating chromium toxicity. The varieties show different levels of tolerance or sensitivity to chromium stress (Fig. S1; Figs. 1a-g). The average germination rates were obtained from Brassica juncea varieties grown under different Cr concentrations. Early raya had the highest germination rate among the tested varieties, reaching 90.33% even at 1000 µM Cr. In contrast, Sindh raya showed the most noticeable reduction, with germination dropping to 56.66% (Figure 1a). The lowest germination was recorded in Sindh Raya and S-9. Based on all growth traits (root, shoot length, and root and shoot fresh and dry weight), these varieties were also most adversely affected, showing marked declines in seedling growth and biomass production (Figs. 1a-g). Furthermore, THB-81 experienced a notable increase in biomass, suggesting that moderate Cr levels (200 µM Cr) could trigger a beneficial physiological response in this variety. The data shown in Fig. 1 indicate that chromium concentrations above 200 µM are toxic to some Brassica juncea varieties, causing significant decreases in root and shoot development, as well as reduced overall plant height. Conversely, some varieties, notably JS-13, Early raya, and THB-81, display some tolerance or even slight benefits at very low Cr levels (10-100 µM). On the other hand, Sindh raya and S-9 are among the most sensitive, exhibiting less vigorous growth and a faster decline in performance as Cr levels increase (Figs.1a-g). Overall, the data in Fig. 1 highlight the genetic differences in how plants respond to heavy metal stress, suggesting that varieties like Early raya and THB-81 should be studied more closely for phytoremediation, particularly in regions with low to moderate chromium pollution. Cr accumulation in Brassica juncea varieties Cr was mostly accumulated in plant roots, with restricted translocation to aboveground tissues (Fig. 2). In particular, at 1000 µM Cr, THB-81 and Early raya accumulated the maximum amount of Cr in their roots, containing 552.28 µg g⁻¹ DW and 429 µg g⁻¹ DW, respectively (Fig. 2a). The accumulation of Cr differed among varieties, but the highest amount of Cr accumulation in the stem was observed in Early raya (88.01 µg g⁻¹ DW) (Fig. 2b). Early raya also accumulated relatively high metal levels far above the toxic concentration in the shoots. Moreover, the lowest Cr uptake and the least amount of translocation to aboveground parts were observed in Sindh raya and S-9, suggesting an exclusion strategy in these varieties (Fig. 2b). The Cr content in leaves differed in varieties, supporting the existence of variety-specific mechanisms for Cr translocation and sequestration (Fig. 2c). Accumulation patterns of Fe, Ca, K, and Mg in different Brassica juncea varieties The results showed that metal accumulation differed among varieties. The overall accumulation patterns varied among varieties, with increasing Cr concentrations. The accumulation of Fe, Ca, Mg, and K in plant tissues changed dramatically when exposed to Cr stress (Figs. 3a-m and Table S2). Figure 3 shows the increase in accumulation of major essential elements (Ca, Mg, K, and Fe) in the plant tissues with a concomitant increase in Cr levels in the media. This aligns with known responses to heavy metal stress, where plants modify their nutrient uptake and transport. The patterns of accumulation differ notably between roots, stems, and leaves, illustrating how various tissues respond to and manage metal stress. Roots typically show the highest metal accumulation, particularly at elevated Cr levels, as they serve as the initial point of contact. As illustrated in Figures 1 and 2, different Brassica juncea varieties respond uniquely—some are better at accumulating or regulating certain metals, which likely accounts for their different levels of Cr tolerance (Fig. 3). For all varieties, leaf Fe content increases with increasing Cr concentration. Ganj sarhen and Sindh raya exhibit particularly high Fe accumulation in their leaves at the maximum Cr level (1000 µM). In contrast, S-9 and THB-81 have the lowest overall Fe levels in their leaves, indicating they may be less effective at transporting Fe to the shoots or that Fe uptake is more restricted (Fig. 3i). Across all varieties, root Fe content significantly surpasses leaf Fe, a common plant strategy to prevent heavy metal toxicity in photosynthetic tissues (Fig. 3g). The highest root Fe accumulation occurs in JS-13, Early raya, and Ganj sarhen at 1000 µM Cr. S-9 shows the lowest root Fe, possibly related to its overall slower growth and greater sensitivity. Although the Fe content in the stem tissues is lower than in roots and leaves, it still increases with high Cr (Fig. 3h). Similarly, Ca content in the leaves increases with an increase in Cr concentrations across most varieties (Figs. 3a, b, c). Interestingly, Ganj sarhen has exceptionally high Ca content in its leaves, peaking at 1000 µM Cr in the media. In contrast, S-9 and Sindh raya show the lowest leaf Ca content, which may reflect a general stress response or less effective nutrient management (Fig. 3c). Root Ca content also increases as Cr levels rise (Fig. 3a). Ganj sarhen and THB-81 display the highest Ca accumulation in their roots, suggesting a strong capacity for Ca uptake and storage at the root level. At the same time, the Ca content in these varieties also increases in stems, indicating efficient translocation from roots to shoots (Fig. 3b). Magnesium (Mg) content in leaves generally rises with increasing Cr levels (Fig. f). Ganj sarhen displays a distinct pattern, maintaining consistently low leaf Mg across all concentrations, unlike other varieties that show increases. This may suggest a different homeostatic regulation. Early raya, JS-13, and THB-81 have the highest leaf Mg accumulation. Stem and root Mg levels also increase with Cr concentration (Figs. 3d, e) in these varieties. In all varieties, root K levels positively correlate with increasing Cr concentration (Figs. 3k, l, m). JS-13 and THB-81 showed the highest K accumulation in roots, indicating that they may absorb and sequester K more effectively in roots as a defense against Cr toxicity. S-9 and Sindh raya varieties accumulated the lowest amount of K in roots across all treatments, suggesting that these varieties are possibly more affected by Cr stress compared to other varieties, indicating their high sensitivity to Cr toxicity (Fig. 3). Leaves generally contain the highest K concentration compared to roots and stems, and K levels increase with rising Cr in all varieties (Fig. 3m). Early raya and JS-13 have the highest leaf K and Ganj sarhen and S-9 show the lowest K in leaves with increase in Cr concentration. Cr may compete with other metal transporters in these varieties, causing shifts in uptake patterns. Cr-tolerant plant varieties might actively increase the absorption of other metals, such as Ca, Mg, and K, either as a stress response or to mitigate Cr toxicity. For example, calcium (Ca) acts as a secondary messenger in stress signaling, while increased iron (Fe) uptake might counteract Cr-induced oxidative stress. Some varieties, such as Ganj sarhen (for Ca) and JS-13 (for Fe and K), show notably high metal accumulation in specific tissues. This buildup, mostly in roots and stems, could prevent Cr from reaching sensitive photosynthetic tissues such as leaves. On one hand, JS-13, Early raya, and THB-81 accumulate potassium (K) significantly, especially under high Cr levels, supporting their tolerance to Cr stress. On the other hand, the S-9 and Ganj sarhen show low potassium accumulation in their leaves. Overall, the data suggests that JS-13, Early raya, and THB-81 are more resilient, likely due to their ability to regulate and store essential metals more effectively under Cr stress, thereby boosting their tolerance. In contrast, S-9 and Sindh raya both seem to be sensitive and are less capable of handling this stress, as shown by their lower metal accumulation. Correlation between Cr and Fe, Ca, K, and Mg uptake Plants grown in metal-enriched media absorb metal ions to varying amounts depending on their uptake capacity and tolerance mechanisms. For this purpose, the accumulator plant, Early raya, and the excluder plant, Sindh raya, were selected to investigate the relationships between Cr and Fe, Mg, K, and Ca (Figs. 4a and b). A regression analysis was conducted between Cr and other elements (Ca, Mg, K, Fe) in the roots of the accumulator, Early raya, and the excluder, Sindh raya. Fe (R² = 0.9614), Mg (R² = 0.9711), and Ca (R² = 0.9154) exhibited the highest positive correlations with Cr accumulation in Early raya. At the same time, K showed a relatively weaker correlation (R² = 0.7371) compared to the other elements (Fig. 4a). Cr accumulation exhibited relatively weak associations with Fe (R² = 0.7994), Mg (R² = 0.8293), K (R² = 0.5740), and Ca (R² = 0.4627) in Sindh raya, implying a lower efficiency in nutrient regulation under Cr stress compared to Early raya (Fig. 4b). These results indicate that the nutrient homeostasis in response to Cr stress in Early raya is more robust than Sindh raya, highlighting varietal differences in Cr affinity. PCA analysis of six Brassica juncea varieties grown on different chromium concentrations Based on chromium accumulation in their tissues, the PCA analysis reveals the distribution of six Brassica juncea varieties (Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81) (Fig. 5). The PCA biplot was generated to evaluate the accumulation and tolerance potential of Brassica juncea varieties exposed to different Cr concentrations. In the PCA analysis, the two components, PC axis 1 and PC axis 2, accounted for 83.1% and 16.5% of the total variance, respectively, resulting in a total cumulative variance of 99.6%. PCA results showed that the varieties fall into three groups according to their Cr accumulation. Here, we aimed to assess the variety-specific selectivity for Cr accumulation. As shown in Figure 5, the biplot displays the distinct Cr accumulation status of six Brassica juncea varieties. Accordingly, a clear distinction is observed between the different Cr treatments across the six studied varieties. As a result, the B. juncea varieties were positioned on the right, middle, and left of the PCA biplot. One group, in green, includes Cr accumulators (Early raya and THB-81), the second, in gray, represents intermediate types (JS 13, Ganj sarhen), and the third, in red, comprises Cr excluders (S-9 and Sindh raya). The separation along PCA1 (which accounts for 83.1% of the variance) particularly emphasizes the contrasting patterns of metal accumulation (Fig. 5). Gene expression in Brassica juncea varieties‒ Early raya and Sindh raya Primers were designed using the mRNA sequences from the GenBank and EMBL databases for the three ROS-related genes Ascorbate Peroxidase (APX), Iron-Superoxide Dismutase (FeSOD), and Respiratory Burst Oxidase Homolog (RBOH) (Table S1), with the ubiquitin gene serving as a reference. Gene expression patterns for enzymes involved in oxidative stress differed between the accumulator Early raya and the excluder Sindh raya. APX, FSD, and RBOH were expressed in Early raya, with their expression levels increasing concomitantly with an increase in Cr. On the other hand, only one gene, APX, was expressed in Sindh raya, with increasing Cr level. A substantial expression of Ascorbate peroxidase (APX) was observed in Sindh raya at 1000 µM Cr (a 13.74-fold increase relative to the control), indicating a Cr concentration-dependent response. The expression of FeSOD and RBOH was slightly altered with increasing Cr levels in the media (see Fig. 6). DISCUSSION This study aimed to find a solution for Cr pollution using environmentally friendly and cost-effective methods, such as phytoremediation, an emerging technology for sustainable solutions to metal-polluted soils (Han et al. 2023 ; Lee et al. 2025 ). In this context, we obtained six Brassica juncea varieties developed by a geneticist in the oilseed department at the agricultural institute in Pakistan, which were chosen for their tolerance to drought and salt stress. We used this genetic resource to identify suitable metal-resistant varieties to address the Cr pollution problem in Turkey. We examined various physiological and molecular parameters to understand how different varieties respond to Cr stress. Our results show that the toxic effects of Cr were partly reduced by the accumulation of specific macro- and micronutrients, and that different genotypes employ distinct strategies for Cr detoxification. Additionally, we identified Cr-tolerant, accumulator, and excluder varieties of Brassica juncea to combat Cr pollution both in Turkey and worldwide. Growth responses and Cr accumulation in different varieties of Brassica juncea Concomitant increases in Cr levels (0–1000 µM) in media negatively affected germination and growth, with Early raya preserving a high germination rate (90.33% at 1000 µM), while Sindh raya showed a significant decline (56.83%). Growth inhibition was most pronounced in Sindh raya and S-9. Furthermore, the Early raya and THB-81 varieties exhibited enhanced tolerance, possibly attributable to efficient detoxification strategies such as chromium sequestration and compartmentalization into metabolically low-active tissues, including the cell wall, leaf trichomes, and vacuoles, as well as high expression of ROS-related genes and enzymes (Wakeel et al. 2020 ; Han et al. 2023 ). Cr has no particular biological role in plant cells and is not necessary for plant growth and development (Oliveira 2012 ; Sharma et al. 2020 ). Several plant species have developed different strategies to overcome Cr toxicity. Data in the present study show that THB-81 sequestered up to 552.28 µg g⁻¹ DW Cr in roots with no toxicity effect. This is likely due to the vacuolar compartmentalization of Cr or high cell wall binding (Shahandeh and Hossner 2000 ; Pulford et al. 2001 ; Han et al. 2023 ; Shourie et al. 2024 ). On the contrary, the S-9 and Sindh raya varieties accumulated very low Cr in their roots and shoots, indicating divergent varietal strategies for metal stress response. The significant Cr accumulation in stems of Early raya (88.01 µg g⁻¹ DW) indicates that Cr is possibly translocated via xylem loading, potentially through non-selective cation channels or sulfur transporters, which are known to facilitate Cr(III) uptake (Prado et al. 2010 ; Gill et al. 2015 ; de Oliveira et al. 2016 ; Zhang et al. 2018 ). On the other hand, Sindh raya showed minimal Cr uptake (7.83 µg g⁻¹ DW in roots), probably due to active efflux, restricted metal influx, and limited translocation to the shoots, which may be due to lignified casparian strips in the root endodermis acting as a barrier to apoplastic movement of Cr. (Yamaji et al. 2013 ) have shown similar results with Zn accumulation in rice. The different varietal responses to Cr exclusion or accumulation are presumably due to the downregulation or upregulation of metal transporters, e.g., HMA4, NRAMP3, or increased chelation of Cr in the rhizosphere caused by high expression of exudate biosynthesis genes rendering Cr in an innocuous form (Clemens 2006 ; Shourie et al. 2024 ). Cr-nutrient interactions Strong correlations were observed between Cr and essential metals, such as Fe, Ca, Mg, and K, in the Cr-tolerant varieties (Early raya and THB-81), suggesting competitive metal uptake and a stress response to cope with Cr stress (Vert et al. 2002 ). Notably, THB-81 had higher Ca levels in its roots (811.36 µg g⁻¹ DW), which could help reduce Cr toxicity by stabilizing membrane integrity and reducing Cr-induced damage (Hirschi 2004 ). In contrast, Cr-sensitive varieties (S9 and Sindh raya) exhibited weaker correlations, indicating that they primarily rely on passive exclusion of Cr. Principal component analysis (PCA) and genetic implications Principal Component Analysis (PCA) was employed to investigate genotype-specific responses to Cr stress and its interaction with other nutrients (Dolezalova et al. 2015 ). In the present study, PCA distinguished Brassica juncea varieties based on their physiological responses to Cr stress, categorizing them into several distinct groups. This classification highlights a variety-specific management of Cr stress, with some accumulating Cr and others excluding it. These varietal differences may reflect different strategies in metal transporter expression, detoxification capacity, or intracellular compartmentalization (Shanker et al. 2005 ). The close relationship between THB-81 and Early raya in the accumulator group indicates that these varieties may share genetic traits that enhance Cr uptake and sequestration, such as duplicated metal transporter genes or improved root cortical aerenchyma (DalCorso et al. 2019 ). Conversely, excluder varieties like S-9 and Sindh raya may carry allelic variations that provide resistance by limiting metal translocation into the xylem. This could occur through thicker exodermal layers or increased activity of FRD3-like efflux transporters (Durrett et al. 2007 ). The hypothesis that these varieties use different strategies for chromium uptake, exclusion, and detoxification is supported by the varying spatial distribution of varieties in the PCA analysis. These findings have important practical implications in the phytoremediation of heavy metals. Accumulator varieties show promise for phytoremediation of Cr-contaminated soils, whereas excluder genotypes might be better suited for safe cultivation in polluted areas. This PCA-based classification provides a useful framework for breeding efforts aimed at enhancing Cr tolerance or reducing metal accumulation in edible plant parts. Distinctive gene expression patterns in Brassica juncea varieties under Cr stress Sindh raya and Early raya showed varied reactions to the gene expression of oxidative-related enzymes when subjected to different levels of Cr. This pronounced varietal response is possibly due to their ability to cope differently with oxidative stress. Exposure of Sindh raya to 1000 µM Cr increased APX (ascorbate peroxidase) expression to 13.74-fold compared to controls. Meanwhile, the levels of FeSOD (Fe-superoxide dismutase) and RBOH (respiratory burst oxidase homolog) remained unchanged despite Cr exposure, suggesting that Sindh raya primarily depends on the APX system for oxidative stress defense. Similar findings were reported by (Jung et al. 2020 ) in oilseed rape seedlings exposed to Cd stress. These findings indicate that the two varieties employ different mechanisms to manage ROS generated by metal toxicity (see Fig. 6 a). In contrast, Early raya displays a more active and cohesive antioxidant defense system. Unlike Sindh raya, all three genes (Fig. 6 b) showed strong induction, particularly at elevated Cr levels. This synchronized response in Early raya suggests a highly flexible strategy against oxidative stress. FeSOD and RBOH expression are crucial in the plant's response system, which is activated during oxidative stress. For example, (Regon et al. 2022 ) demonstrated that these genes are crucial for aromatic Keteki Joha rice to adapt to oxidative stress caused by ferrous iron (Fe²⁺). The activation of these ROS-related genes by heavy metals or other abiotic stresses suggests that Early raya has developed a more comprehensive mechanism to handle metal stress (Wang et al. 2008 ; Małecka et al. 2019 ). Practical implications for phytoremediation strategies Our data in Table 1 provides a clear, evidence-based framework for selecting the most suitable plant variety to achieve a specific environmental goal, rather than relying on a generic approach (del Real et al. 2013 ). Here, Early raya and THB-81 are grouped as Cr accumulators, which tolerate Cr toxicity through Mg accumulation, while S-9 and Sindh raya were shown to be strong excluders of Cr. Table 1 The Brassica juncea varieties are tentatively classified into six groups, considering the influence of other essential elements on Cr uptake and translocation Early raya Strong accumulator Co-accumulates Mg, maybe stress-tolerant via internal detoxification THB-81 Accumulator (esp. at high Cr) Possibly tolerates via moderate Mg accumulation JS-13 Moderate accumulator Fe uptake seems unrelated to Cr S-9 Strong excluder Cr-resistant, may avoid uptake or sequester in roots Sindh raya Strong excluder Cr-resistant, similar to S-9 Ganj sarhen Weak Cr association, high Ca Likely Ca-mediated defense strategy The different metal strategies studied here can guide future plant breeding and genetic engineering efforts. As shown in Table 1 , pinpointing specific mechanisms—such as Ca-mediated defense, Mg-mediated tolerance, or Cr-opposed uptake—lays a foundation for creating new plant varieties optimized for phytoremediation or food safety. This is a significant and unique finding regarding how different varieties respond to Cr and how it interacts with other essential elements in plants. These varieties could enable safe cultivation in areas contaminated with metal pollutants. CONCLUSION Given that Turkey is one of the world’s leading chromium exporters and that various regions, particularly regions (i.e., Usak, Manisa, İzmir, Diyarbakir) of Anatolia, suffer from extensive Cr contamination due to leather tanning and mining activities. The findings of this study hold substantial environmental and agricultural significance for safe agriculture and food, not only for Turkey but also for other countries facing similar challenges. This research will be important in addressing global environmental issues. In this context, we explored solutions for alleviating the Cr pollution problem by using suitable plant varieties for remediation or safe cultivation in metal-polluted soils. Our results grouped the Brassica juncea varieties into three categories: the first group, Cr accumulators (Early raya and THB-81); the second group, intermediate types (JS-13 and Ganj sarhen); and the third group, excluders (S-9 and Sindh raya). We propose that Early raya and THB-81 varieties could be used for phytoremediation of Cr-contaminated sites. In contrast, Sindh raya and S-9 (excluders) could be cultivated for safe agriculture in polluted areas, thereby reducing Cr uptake into the food supply. These variations in Cr uptake, translocation, and accumulation across varieties offer important insights to local authorities, agronomists, and breeders in Turkey and beyond. This helps in developing customized, sustainable approaches to tackle chromium pollution in agriculture. Declarations Acknowledgements We thank the Agricultural Research Institute for providing us with Brassica juncea L. seeds. Funding This study was financially supported by the Council of Higher Education (YOK) through the Uşak University Research Grant (BAP) under project number 2024/MF002 to Prof. Dr. Abdulrezzak Memon and Prof. Dr. Ahmet Kahraman. Author Contributions NM performed most of the experiments, while ARM and HA assisted with some of them. NM and ARM wrote the manuscript, with contributions from all authors. NM, ARM, HA, BK, LAB, and AK interpreted the data. ARM designed and supervised the project. All authors have read and agreed to the published version of the manuscript. Ethics approval: Not applicable Consent to participate: Not applicable. Consent for publication: Not applicable. Clinical trial number: Not applicable. Competing interests: The authors declare that they have no competing interests. Data Availability All data generated or analyzed during this study are included in this article and its supplementary information files. 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Plant physiology 162 (2):927-939 Zhang X, Kang J, Pang H, Niu L, Lv J (2018) Sulfur mediated improved thiol metabolism, antioxidant enzymes system and reduced chromium accumulation in oilseed rape (Brassica napus L.) shoots. Environmental Science and Pollution Research 25 (35):35492-35500 Zulfiqar U, Haider FU, Ahmad M, Hussain S, Maqsood MF, Ishfaq M, Shahzad B, Waqas MM, Ali B, Tayyab MN (2023) Chromium toxicity, speciation, and remediation strategies in soil-plant interface: A critical review. Frontiers in Plant Science 13:1081624 Supplementary Files Suppl.Material1.docx Supplementary Information Table S1 Forward and reverse primers for ubiquitin, APX, RBOH, and FeSOD in Brassica juncea were designed using Primer3 and validated with the BLAST tool. Table S2 P-values for the interactions of Cr with Ca, Fe, Mg, and K in the roots of Brassica juncea varieties under Cr stress Fig. S1 Morphological response of Brassica juncea L. var. Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81 seedlings were exposed to different concentrations of chromium (Cr). Plants were grown under 0, 10, 50, 100, 200, 500, and 1000 µM Cr for two weeks (15 days). Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revision 14 Oct, 2025 Reviewers agreed at journal 16 Aug, 2025 Reviewers invited by journal 15 Aug, 2025 Editor invited by journal 14 Aug, 2025 Editor assigned by journal 13 Aug, 2025 First submitted to journal 08 Aug, 2025 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-7303811","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501159960,"identity":"8ce234fa-7352-41cf-9fb1-0755b7c447f0","order_by":0,"name":"Nuriye Merakli","email":"","orcid":"","institution":"Department of Molecular Biology and Genetics, Faculty of Engineering and Life Sciences, Usak University, Usak Merkez, 64200 Usak","correspondingAuthor":false,"prefix":"","firstName":"Nuriye","middleName":"","lastName":"Merakli","suffix":""},{"id":501159961,"identity":"7f306053-9278-4cfb-9847-3a56222e6835","order_by":1,"name":"Abdulrezzak Memon","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-9447-6453","institution":"Department of Molecular Biology and Genetics, Faculty of Engineering and Life Sciences, Usak University, Usak Merkez, 64200 Usak","correspondingAuthor":true,"prefix":"","firstName":"Abdulrezzak","middleName":"","lastName":"Memon","suffix":""},{"id":501159962,"identity":"77593d7e-b836-47f8-9929-6071e4bd6480","order_by":2,"name":"Huseyin Altundag","email":"","orcid":"","institution":"Department of Chemistry, Faculty of Science, Sakarya University, Sakarya","correspondingAuthor":false,"prefix":"","firstName":"Huseyin","middleName":"","lastName":"Altundag","suffix":""},{"id":501159963,"identity":"2d6ab735-c986-4656-9150-78990a86428e","order_by":3,"name":"Baris Kaki","email":"","orcid":"","institution":"Department of Animal Science, Faculty of Agriculture, Usak University, Usak Merkez, 64200 Usak","correspondingAuthor":false,"prefix":"","firstName":"Baris","middleName":"","lastName":"Kaki","suffix":""},{"id":501159964,"identity":"329c847a-5a53-4726-885c-4b9d5fdb8ec1","order_by":4,"name":"Liaquat Ali Bhutto","email":"","orcid":"","institution":"Director, Agriculture Research Center, Tandojam, Pakistan","correspondingAuthor":false,"prefix":"","firstName":"Liaquat","middleName":"Ali","lastName":"Bhutto","suffix":""},{"id":501159965,"identity":"e220358b-a3da-4f4a-b888-41aa6cd1a067","order_by":5,"name":"Ahmet Kahraman","email":"","orcid":"","institution":"Department of Molecular Biology and Genetics, Faculty of Engineering and Life Sciences, Usak University, Usak Merkez, 64200 Usak","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Kahraman","suffix":""}],"badges":[],"createdAt":"2025-08-05 20:19:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7303811/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7303811/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89821265,"identity":"f3d29aaa-0fbb-461d-8b8d-3c03d475deab","added_by":"auto","created_at":"2025-08-25 11:38:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":443371,"visible":true,"origin":"","legend":"\u003cp\u003eshows germination rate (a), plant height (b), root length (c), root fresh weight (d), root dry weight (e), shoot fresh weight (f), and shoot dry weight (g) of \u003cem\u003eBrassica juncea\u003c/em\u003e varieties- Early raya, Ganj sarhen, JS-13, Sindh raya, S-9, and THB-81 grown at different Cr concentrations (0-1000 µM). Data are shown as ± standard deviations (SD) (n = 3). Grouping (a-g) was performed using Tukey’s test. Different letters indicate significant differences, highlighting varietal responses to Cr.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/c150d13c657950c4745817e4.png"},{"id":89821264,"identity":"4a2bb76c-582f-4b71-a8d6-405481d4f8f2","added_by":"auto","created_at":"2025-08-25 11:38:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133367,"visible":true,"origin":"","legend":"\u003cp\u003eChromium in different tissues of \u003cem\u003eBrassica juncea\u003c/em\u003e varieties (µg g⁻¹ DW). The accumulation of Cr (µg g⁻¹ DW) in the roots (a), stems (b), and leaves (c) of \u003cem\u003eBrassica juncea \u003c/em\u003evarieties- JS-13, Early raya, Sindh raya, S-9, Ganj sarhen, and THB-81 treated with different concentrations (0, 10, 50, 100, 200, 500, and 1000 µM) of CrCl₃ for two weeks (15 days). Data are means ± standard deviations (SD) (n = 3), with values indicating significant differences from the control at p \u0026lt; 0.05. Different letters indicate significant differences, highlighting varietal responses to Cr.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/5c795cedb4c71d994e829c97.png"},{"id":89821266,"identity":"eb44668a-3fdd-436a-8b3a-871a56687569","added_by":"auto","created_at":"2025-08-25 11:38:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":702013,"visible":true,"origin":"","legend":"\u003cp\u003eCa (a, b, c), Mg (d, e, f), Fe (g, h, i), and K (k, l, m) distribution in different tissues of \u003cem\u003eBrassica juncea \u003c/em\u003evarieties. The accumulation of metals (µg g⁻¹ DW) in roots, stems, and leaves of \u003cem\u003eBrassica juncea \u003c/em\u003evarieties- JS-13, Early raya, Sindh raya, S-9, Ganj sarhen, and THB-81 plants, treated with CrCl₃ at different concentrations for two weeks (15 days). Data are means ± standard deviations (SD) (n = 3), with values indicating significant differences from the control at p\u0026lt; 0.001. Different letters indicate significant differences, highlighting varietal responses to Cr.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/49cbc756458b349ca8745f89.png"},{"id":89821295,"identity":"fbc8de15-3f2a-46c6-b028-2a7b78a33d61","added_by":"auto","created_at":"2025-08-25 11:38:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183404,"visible":true,"origin":"","legend":"\u003cp\u003eRegression analysis between Cr and Ca, Fe, Mg, and K contents (µg g⁻¹ DW) in the roots of \u003cem\u003eBrassica juncea\u003c/em\u003e Early raya (a) and Sindh raya (b) under chromium stress.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/b58716ad47cbf41036d6a138.png"},{"id":89821282,"identity":"bb11097e-f80f-4f0a-864d-b00684431445","added_by":"auto","created_at":"2025-08-25 11:38:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85410,"visible":true,"origin":"","legend":"\u003cp\u003eA PCA biplot showing Cr accumulator and excluder varieties of \u003cem\u003eBrassica juncea\u003c/em\u003e. The total variance explained by PC1 and PC2 is 99.6%, indicating strong differentiation between varieties.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/ed7c9df80f8d533f95716672.png"},{"id":89821283,"identity":"7a2bb888-612a-4925-bbc6-af92b8e4ec0c","added_by":"auto","created_at":"2025-08-25 11:38:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":91985,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of Ascorbate Peroxidase (APX), Iron-Superoxide Dismutase (FeSOD), and Respiratory Burst Oxidase Homolog (RBOH) in \u003cem\u003eBrassica juncea\u003c/em\u003e var. Early raya (a) and Sindh raya (b) grew at different levels of Cr.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/ce06fdc17bb0ad6d85ae8bc7.png"},{"id":89821989,"identity":"78ce445c-f6f4-438f-a611-ac41d37e4ac6","added_by":"auto","created_at":"2025-08-25 11:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2358017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/e54956ec-0297-44a5-9ca2-a723bb1e416e.pdf"},{"id":89821271,"identity":"4d78a409-184f-4d37-a873-34a08790e65c","added_by":"auto","created_at":"2025-08-25 11:38:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1700627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1\u003c/strong\u003e Forward and reverse primers for ubiquitin, APX, RBOH, and FeSOD in \u003cem\u003eBrassica juncea\u003c/em\u003ewere designed using Primer3 and validated with the BLAST tool.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2\u003c/strong\u003e P-values for the interactions of Cr with Ca, Fe, Mg, and K in the roots of \u003cem\u003eBrassica juncea\u003c/em\u003evarieties under Cr stress\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1\u003c/strong\u003e Morphological response of \u003cem\u003eBrassica juncea \u003c/em\u003eL. var. Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81 seedlings were exposed to different concentrations of chromium (Cr). Plants were grown under 0, 10, 50, 100, 200, 500, and 1000 µM Cr for two weeks (15 days).\u003c/p\u003e","description":"","filename":"Suppl.Material1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7303811/v1/f474750152eb6b12ae8a1797.docx"}],"financialInterests":"","formattedTitle":"Phytoremediation Potential and Differential Chromium (Cr) Accumulation and Gene Expression in Brassica juncea Varieties for Contaminated Soils in Turkey","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, there has been significant development activity involving industrialization and rapid urbanization across nearly all of Turkey. The leather and textile industries are among the sectors that have experienced considerable growth. In many parts of Turkey, the environment has reached or exceeded its carrying capacity regarding soil and water pollutants such as lime (Ca(OH)₂), sodium sulfide (Na₂S), sodium chloride (NaCl), various dyes, and toxic heavy metals, especially chromium (Cr), released by these industrial sectors (Harmancioglu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eChromium (Cr) is one of the toxic heavy metals that can harm soil, groundwater, and ecosystems due to its widespread and unconscious use in industrial processes (Ertani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Adnan et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In Turkey, several regions, especially Anatolia, have significant mining and leather tanning activities, most of which generate waste effluent that is a major source of soil and river water pollution (Akcay et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Dundar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tokatli et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These activities negatively impact agriculture, animal husbandry, and the quality of drinking water by contaminating soil and rivers. Turkey's most polluted rivers include the Euphrates, Tigris, Gediz, and Menderes; although they are vital for irrigation, they pose serious risks to public health and food safety (Aydin and Kucuksezgin \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Şent\u0026uuml;rk and Yıldız \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The increasing industrial activity continues to elevate environmental pollution, with long-lasting effects on regional ecosystems (Saxena \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As industrial use of chromium grows, its presence as an environmental contaminant has increased significantly (Coetzee et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eChromium lacks any essential function in plant metabolism; however, it exerts widespread phytotoxic effects that impair nearly all biological functions in plants (Shahid et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cr can interfere with plants' ability to absorb and maintain the balance of vital mineral elements. Competition with other elements interferes with physiological and metabolic functions, leading to growth suppression, oxidative damage, chlorosis, root impairment, and nutritional imbalance, which can ultimately result in plant mortality (Anjum et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh and Malaviya \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). One of the most crucial consequences of Cr stress is the impairment of antioxidant defense systems and the disturbance of the equilibrium between the generation of reactive oxygen species (ROS), which cause cellular damage (Samantary \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Saud et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eChromium contamination poses a serious threat to global food security by impairing the growth and yield of essential crops, including vegetables, cereals, and legumes (Vasilachi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To reduce its harmful effects, it is crucial to investigate how chromium behaves in soil environments, assess its toxic impact on plant growth and metabolism, and develop effective, long-term remediation strategies. Among the benefits these activities can provide are improving soil quality and encouraging environmental sustainability (Xia et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recent research has focused on how crop plants absorb and accumulate chromium and how this process may affect human health through the food chain (del Real et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zulfiqar et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phytoremediation, the use of plants to extract, sequester, or detoxify pollutants, stands out as a sustainable and efficient method to clean up environments contaminated with heavy metals (Memon \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Doğru et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Enhancing the phytoremediation capacity of plants requires a thorough understanding of the physiological and molecular processes that control the uptake of metals, their interaction with essential nutrients, and the mechanisms involved in detoxification (Memon and Schr\u0026ouml;der \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, identifying and utilizing promising plant genotypes that can either accumulate or exclude the target metal is crucial for successful phytoremediation.\u003c/p\u003e\u003cp\u003eGrown for both agricultural and industrial purposes worldwide, \u003cem\u003eBrassica juncea\u003c/em\u003e L. (Brown mustard), is an important edible oilseed crop with significant nutritional and economic value. It is also recognized for its strong ability and significant potential in phytoremediation, especially in areas affected by environmental pollution such as heavy metal-contaminated soils (Anjum et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Due to its reported tolerance to several heavy metals, \u003cem\u003eB. juncea\u003c/em\u003e is considered a sustainable and environmentally friendly option for soil remediation (Singh et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Finding and selecting genotypes with natural tolerance to heavy metals is an effective way to develop improved varieties suitable for growing in areas contaminated with heavy metals (del Real et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, a notable gap remains in understanding \u003cem\u003eB. juncea\u003c/em\u003e genotypes that can be adapted to Turkey\u0026rsquo;s specific climate conditions and provide efficient Cr control under field-related stress levels.\u003c/p\u003e\u003cp\u003eThis study was conducted to investigate the capacity of six \u003cem\u003eBrassica juncea\u003c/em\u003e varieties, Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81, to accumulate and tolerate chromium (Cr) at different concentrations (0, 10, 50, 100, 200, 500, and 1000 \u0026micro;M). The study also explores the potential roles of key elements like Ca, Fe, K, and Mg in reducing Cr toxicity. To understand the molecular mechanisms that boost antioxidant defenses under chromium stress, the transcription levels of ROS-related genes\u0026mdash;ascorbate peroxidase (APX), respiratory burst oxidase homolog (RBOH), and iron superoxide dismutase (FeSOD)\u0026mdash;were assessed via RT-qPCR in the root tissues of the Cr-accumulator variety (Early raya) and the Cr-excluder variety (Sindh raya).\u003c/p\u003e\u003cp\u003eOur work offers an important and practical, comprehensive, two-pronged approach for addressing contaminated sites polluted with Cr. One approach utilizes accumulator varieties for active soil remediation, whereas the other employs excluder varieties such as Sindh raya for sustainable and safe farming. This innovative, multi-varietal comparison of chromium phytoremediation provides a more thorough and practical approach to managing soil contamination, addressing cleanup, food safety, and developing effective and safe strategies for metal-contaminated soils.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePlant material and experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeeds of \u003cem\u003eBrassica juncea\u003c/em\u003e (JS-13, Early raya, Sindh raya, S-9, Ganj sarhen, and THB-81) were obtained from the Agricultural Research Institute, Tandojam, Pakistan. Seeds were surface-sterilized with 5% sodium hypochlorite and Tween-20 for 15 min, then rinsed with distilled water. The plant growth conditions and cultivation methods are the same as described previously (Meraklı et al. 2022). Seeds (100 per pot) were sown in vermiculite and germinated under controlled conditions (25 \u0026plusmn; 2\u0026deg;C day/20 \u0026plusmn; 2\u0026deg;C night, 16 h light/8 h dark, 150 \u0026mu;mol/m\u0026sup2;/s light intensity, 60 \u0026plusmn; 5% humidity). On day 5, germinated seeds (radicle \u0026ge; 2 mm) were counted, and the germination rate (%) was calculated according to (Akinci and Akinci 2010). Seedlings were treated with CrCl₃\u0026middot;6H₂O at 0, 10, 50, 100, 200, 500, and 1000 \u0026mu;M in 1/4-strength Hoagland solution (Hoagland and Arnon 1938). Control plants received only the nutrient solution. After two weeks, seedlings were harvested, and roots were rinsed with sterile deionized water. The experiment was performed in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGrowth parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants were harvested after 15 days (two weeks), carefully removed from the pots, washed to remove vermiculite residue, and separated into leaves, stems, and roots. Root and plant lengths were measured, and the fresh weight (FW) of roots and shoots was recorded after harvesting. Plant and root lengths were measured using a meter scale. The samples were oven-dried at 72\u0026deg;C for approximately 48 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetal analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDried plant samples (0.5 g) were digested in a mixture of nitric and perchloric acid (HNO₃: HClO₄, 5:2 v/v) on a hot plate, then filtered through Whatman No. 42 (0.25 \u0026micro;m) and diluted to 10 ml with Milli-Q water (Memon et al. 1979). Metal concentrations were determined using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES; SPECTRO ARCOS) (Meraklı et al. 2022). Chromium standards (Sigma-Aldrich, Germany) and blank samples were prepared according to the method described by (Meraklı et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the roots of control and Cr-treated plants using the RNeasy Plant Kit (Thermo Fisher Scientific) following the manufacturer\u0026rsquo;s protocol. To eliminate genomic DNA contamination, the RNA samples were treated with RNase-Free DNase. RNA concentrations were measured using a NanoDrop spectrophotometer. cDNA synthesis was performed using the Bio-Rad iScript cDNA Synthesis Kit. The reaction mixture contained iScript reaction mix, reverse transcriptase, the RNA template, and nuclease-free water. Quantitative real-time PCR (qRT-PCR) was performed using the LightCycler 480 system (Roche, Germany) with SYBR Green Master Mix. The reaction included gene-specific primers for APX, FeSOD, RBOH, and ubiquitin. The qRT-PCR procedure followed standard protocols (Meraklı and Memon 2023). Gene-specific primers (see Table S1) were designed based on mRNA sequences obtained from the GenBank and EMBL databases. The ubiquitin gene served as the reference gene. Gene expression quantification was performed using the \u0026Delta;Ct method, where \u0026Delta;Ct values are inversely proportional to the initial template amount (Applied Biosystems). Relative gene expression levels were calculated using the 2^\u0026minus;\u0026Delta;\u0026Delta;Ct method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData (mean \u0026plusmn; SD) were analyzed using SPSS (24.0) and Microsoft Excel. Tukey\u0026apos;s test was used to evaluate differences in plant growth, and simple correlation coefficients were used to assess the significance of metal concentrations. Data normality was assessed using the Shapiro-Wilk test, and variance homogeneity was examined using Levene\u0026apos;s test. A three-way ANOVA was used to investigate the effects of the factors and their interactions. Post hoc comparisons were performed using Duncan\u0026apos;s test. Principal Component Analysis (PCA) was performed to identify patterns of Cr accumulation and better understand the relationships among the studied variables. The principal components that explained most of the total variance were extracted, and the distribution of samples was assessed based on Cr accumulation levels. The positioning of the different factors was visualized using biplot graphs, and the loadings of the principal components were interpreted to elucidate their contribution to the observed variation. R (version 4.2.2), Python (version 3.10.0), and SPSS (version 24.0) were used for the analyses, and p \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eEffects of Chromium (Cr) on growth in \u003cem\u003eBrassica juncea\u003c/em\u003e varieties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Bar charts in Fig. 1 illustrate how Cr influences various growth metrics across six \u003cem\u003eBrassica juncea\u003c/em\u003e varieties: Early raya, Ganj sarhen, JS-13, Sindh raya, S-9, and THB-81 grown at different chromium concentrations (0, 10, 50, 100, 500, and 1000 \u0026micro;M). These metrics include seed germination, plant height, root length, root fresh weight, root dry weight, shoot fresh weight, and shoot dry weight (Fig. S1; Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcross all varieties and parameters, a clear dose-dependent response to increasing chromium levels is evident. Low Cr concentrations (10, 50, 100 \u0026micro;M) generally have a stimulating or neutral effect on plant growth compared to the control (0 \u0026micro;M). However, as Cr levels rise to 200, 500, and especially 1000 \u0026micro;M, all growth measurements significantly decrease across all varieties, indicating chromium toxicity. The varieties show different levels of tolerance or sensitivity to chromium stress (Fig. S1; Figs. 1a-g). The average germination rates were obtained from \u003cem\u003eBrassica juncea\u003c/em\u003e varieties grown under different Cr concentrations. Early raya had the highest germination rate among the tested varieties, reaching 90.33% even at 1000 \u0026micro;M Cr. In contrast, Sindh raya showed the most noticeable reduction, with germination dropping to 56.66% (Figure 1a). The lowest germination was recorded in Sindh Raya and S-9. Based on all growth traits (root, shoot length, and root and shoot fresh and dry weight), these varieties were also most adversely affected, showing marked declines in seedling growth and biomass production (Figs. 1a-g). Furthermore, THB-81 experienced a notable increase in biomass, suggesting that moderate Cr levels (200 \u0026micro;M Cr) could trigger a beneficial physiological response in this variety. The data shown in Fig. 1 indicate that chromium concentrations above 200 \u0026micro;M are toxic to some \u003cem\u003eBrassica juncea\u003c/em\u003e varieties, causing significant decreases in root and shoot development, as well as reduced overall plant height. Conversely, some varieties, notably JS-13, Early raya, and THB-81, display some tolerance or even slight benefits at very low Cr levels (10-100 \u0026micro;M). On the other hand, Sindh raya and S-9 are among the most sensitive, exhibiting less vigorous growth and a faster decline in performance as Cr levels increase (Figs.1a-g). Overall, the data in Fig. 1 highlight the genetic differences in how plants respond to heavy metal stress, suggesting that varieties like Early raya and THB-81 should be studied more closely for phytoremediation, particularly in regions with low to moderate chromium pollution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCr accumulation in \u003cem\u003eBrassica juncea\u003c/em\u003e varieties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCr was mostly accumulated in plant roots, with restricted translocation to aboveground tissues (Fig. 2). In particular, at 1000 \u0026micro;M Cr, THB-81 and Early raya accumulated the maximum amount of Cr in their roots, containing 552.28 \u0026micro;g g⁻\u0026sup1; DW and 429 \u0026micro;g g⁻\u0026sup1; DW, respectively (Fig. 2a). The accumulation of Cr differed among varieties, but the highest amount of Cr accumulation in the stem was observed in Early raya (88.01 \u0026micro;g g⁻\u0026sup1; DW) (Fig. 2b). Early raya also accumulated relatively high metal levels far above the toxic concentration in the shoots. Moreover, the lowest Cr uptake and the least amount of translocation to aboveground parts were observed in Sindh raya and S-9, suggesting an exclusion strategy in these varieties (Fig. 2b). The Cr content in leaves differed in varieties, supporting the existence of variety-specific mechanisms for Cr translocation and sequestration (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccumulation patterns of Fe, Ca, K, and Mg in different \u003cem\u003eBrassica juncea\u003c/em\u003e varieties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that metal accumulation differed among varieties. The overall accumulation patterns varied among varieties, with increasing Cr concentrations. The accumulation of Fe, Ca, Mg, and K in plant tissues changed dramatically when exposed to Cr stress (Figs. 3a-m and Table S2).\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the increase in accumulation of major essential elements (Ca, Mg, K, and Fe) in the plant tissues with a concomitant increase in Cr levels in the media. This aligns with known responses to heavy metal stress, where plants modify their nutrient uptake and transport. The patterns of accumulation differ notably between roots, stems, and leaves, illustrating how various tissues respond to and manage metal stress. Roots typically show the highest metal accumulation, particularly at elevated Cr levels, as they serve as the initial point of contact. As illustrated in Figures 1 and 2, different \u003cem\u003eBrassica juncea\u0026nbsp;\u003c/em\u003evarieties respond uniquely\u0026mdash;some are better at accumulating or regulating certain metals, which likely accounts for their different levels of Cr tolerance (Fig. 3). For all varieties, leaf Fe content increases with increasing Cr concentration. Ganj sarhen and Sindh raya exhibit particularly high Fe accumulation in their leaves at the maximum Cr level (1000 \u0026micro;M). In contrast, S-9 and THB-81 have the lowest overall Fe levels in their leaves, indicating they may be less effective at transporting Fe to the shoots or that Fe uptake is more restricted (Fig. 3i). Across all varieties, root Fe content significantly surpasses leaf Fe, a common plant strategy to prevent heavy metal toxicity in photosynthetic tissues (Fig. 3g). The highest root Fe accumulation occurs in JS-13, Early raya, and Ganj sarhen at 1000 \u0026micro;M Cr. S-9 shows the lowest root Fe, possibly related to its overall slower growth and greater sensitivity. Although the Fe content in the stem tissues is lower than in roots and leaves, it still increases with high Cr (Fig. 3h). Similarly, Ca content in the leaves increases with an increase in Cr concentrations across most varieties (Figs. 3a, b, c). Interestingly, Ganj sarhen has exceptionally high Ca content in its leaves, peaking at 1000 \u0026micro;M Cr in the media. In contrast, S-9 and Sindh raya show the lowest leaf Ca content, which may reflect a general stress response or less effective nutrient management (Fig. 3c). Root Ca content also increases as Cr levels rise (Fig. 3a). Ganj sarhen and THB-81 display the highest Ca accumulation in their roots, suggesting a strong capacity for Ca uptake and storage at the root level. At the same time, the Ca content in these varieties also increases in stems, indicating efficient translocation from roots to shoots (Fig. 3b). Magnesium (Mg) content in leaves generally rises with increasing Cr levels (Fig. f). Ganj sarhen displays a distinct pattern, maintaining consistently low leaf Mg across all concentrations, unlike other varieties that show increases. This may suggest a different homeostatic regulation. Early raya, JS-13, and THB-81 have the highest leaf Mg accumulation. Stem and root Mg levels also increase with Cr concentration (Figs. 3d, e) in these varieties. In all varieties, root K levels positively correlate with increasing Cr concentration (Figs. 3k, l, m). JS-13 and THB-81 showed the highest K accumulation in roots, indicating that they may absorb and sequester K more effectively in roots as a defense against Cr toxicity. S-9 and Sindh raya varieties accumulated the lowest amount of K in roots across all treatments, suggesting that these varieties are possibly more affected by Cr stress compared to other varieties, indicating their high sensitivity to Cr toxicity (Fig. 3). Leaves generally contain the highest K concentration compared to roots and stems, and K levels increase with rising Cr in all varieties (Fig. 3m). Early raya and JS-13 have the highest leaf K and Ganj sarhen and S-9 show the lowest K in leaves with increase in Cr concentration. Cr may compete with other metal transporters in these varieties, causing shifts in uptake patterns. Cr-tolerant plant varieties might actively increase the absorption of other metals, such as Ca, Mg, and K, either as a stress response or to mitigate Cr toxicity. For example, calcium (Ca) acts as a secondary messenger in stress signaling, while increased iron (Fe) uptake might counteract Cr-induced oxidative stress. Some varieties, such as Ganj sarhen (for Ca) and JS-13 (for Fe and K), show notably high metal accumulation in specific tissues. This buildup, mostly in roots and stems, could prevent Cr from reaching sensitive photosynthetic tissues such as leaves. On one hand, JS-13, Early raya, and THB-81 accumulate potassium (K) significantly, especially under high Cr levels, supporting their tolerance to Cr stress. On the other hand, the S-9 and Ganj sarhen show low potassium accumulation in their leaves. Overall, the data suggests that JS-13, Early raya, and THB-81 are more resilient, likely due to their ability to regulate and store essential metals more effectively under Cr stress, thereby boosting their tolerance. In contrast, S-9 and Sindh raya both seem to be sensitive and are less capable of handling this stress, as shown by their lower metal accumulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation between Cr and Fe, Ca, K, and Mg uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePlants grown in metal-enriched media absorb metal ions to varying amounts depending on their uptake capacity and tolerance mechanisms. For this purpose, the accumulator plant, Early raya, and the excluder plant, Sindh raya, were selected to investigate the relationships between Cr and Fe, Mg, K, and Ca (Figs. 4a and b). A regression analysis was conducted between Cr and other elements (Ca, Mg, K, Fe) in the roots of the accumulator, Early raya, and the excluder, Sindh raya. Fe (R\u0026sup2; = 0.9614), Mg (R\u0026sup2; = 0.9711), and Ca (R\u0026sup2; = 0.9154) exhibited the highest positive correlations with Cr accumulation in Early raya. At the same time, K showed a relatively weaker correlation (R\u0026sup2; = 0.7371) compared to the other elements (Fig. 4a). Cr accumulation exhibited relatively weak associations with Fe (R\u0026sup2; = 0.7994), Mg (R\u0026sup2; = 0.8293), K (R\u0026sup2; = 0.5740), and Ca (R\u0026sup2; = 0.4627) in Sindh raya, implying a lower efficiency in nutrient regulation under Cr stress compared to Early raya (Fig. 4b). These results indicate that the nutrient homeostasis in response to Cr stress in Early raya is more robust than Sindh raya, highlighting varietal differences in Cr affinity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCA analysis of six \u003cem\u003eBrassica juncea\u003c/em\u003e varieties grown on different chromium concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on chromium accumulation in their tissues, the PCA analysis reveals the distribution of six \u003cem\u003eBrassica juncea\u003c/em\u003e varieties (Early raya, JS-13, Sindh raya, S-9, Ganj sarhen, and THB-81) (Fig. 5). The PCA biplot was generated to evaluate the accumulation and tolerance potential of \u003cem\u003eBrassica juncea\u003c/em\u003e varieties exposed to different Cr concentrations. In the PCA analysis, the two components, PC axis 1 and PC axis 2, accounted for 83.1% and 16.5% of the total variance, respectively, resulting in a total cumulative variance of 99.6%. PCA results showed that the varieties fall into three groups according to their Cr accumulation. Here, we aimed to assess the variety-specific selectivity for Cr accumulation. As shown in Figure 5, the biplot displays the distinct Cr accumulation status of six \u003cem\u003eBrassica juncea\u0026nbsp;\u003c/em\u003evarieties. \u0026nbsp;Accordingly, a clear distinction is observed between the different Cr treatments across the six studied varieties. As a result, the \u003cem\u003eB. juncea\u003c/em\u003e varieties were positioned on the right, middle, and left of the PCA biplot. One group, in green, includes Cr accumulators (Early raya and THB-81), the second, in gray, represents intermediate types (JS 13, Ganj sarhen), and the third, in red, comprises Cr excluders (S-9 and Sindh raya). The separation along PCA1 (which accounts for 83.1% of the variance) particularly emphasizes the contrasting patterns of metal accumulation (Fig. 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression in \u003cem\u003eBrassica juncea\u0026nbsp;\u003c/em\u003evarieties‒ Early raya and Sindh raya\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrimers were designed using the mRNA sequences from the GenBank and EMBL databases for the three ROS-related genes Ascorbate Peroxidase (APX), Iron-Superoxide Dismutase (FeSOD), and Respiratory Burst Oxidase Homolog (RBOH) (Table S1), with the ubiquitin gene serving as a reference. Gene expression patterns for enzymes involved in oxidative stress differed between the accumulator Early raya and the excluder Sindh raya. APX, FSD, and RBOH were expressed in Early raya, with their expression levels increasing concomitantly with an increase in Cr. On the other hand, only one gene, APX, was expressed in Sindh raya, with increasing Cr level. A substantial expression of Ascorbate peroxidase (APX) was observed in Sindh raya at 1000 \u0026micro;M Cr (a 13.74-fold increase relative to the control), indicating a Cr concentration-dependent response. The expression of FeSOD and RBOH was slightly altered with increasing Cr levels in the media (see Fig. 6).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to find a solution for Cr pollution using environmentally friendly and cost-effective methods, such as phytoremediation, an emerging technology for sustainable solutions to metal-polluted soils (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In this context, we obtained six \u003cem\u003eBrassica juncea\u003c/em\u003e varieties developed by a geneticist in the oilseed department at the agricultural institute in Pakistan, which were chosen for their tolerance to drought and salt stress. We used this genetic resource to identify suitable metal-resistant varieties to address the Cr pollution problem in Turkey. We examined various physiological and molecular parameters to understand how different varieties respond to Cr stress. Our results show that the toxic effects of Cr were partly reduced by the accumulation of specific macro- and micronutrients, and that different genotypes employ distinct strategies for Cr detoxification. Additionally, we identified Cr-tolerant, accumulator, and excluder varieties of \u003cem\u003eBrassica juncea\u003c/em\u003e to combat Cr pollution both in Turkey and worldwide.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth responses and Cr accumulation in different varieties of\u003c/b\u003e \u003cb\u003eBrassica juncea\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConcomitant increases in Cr levels (0\u0026ndash;1000 \u0026micro;M) in media negatively affected germination and growth, with Early raya preserving a high germination rate (90.33% at 1000 \u0026micro;M), while Sindh raya showed a significant decline (56.83%). Growth inhibition was most pronounced in Sindh raya and S-9. Furthermore, the Early raya and THB-81 varieties exhibited enhanced tolerance, possibly attributable to efficient detoxification strategies such as chromium sequestration and compartmentalization into metabolically low-active tissues, including the cell wall, leaf trichomes, and vacuoles, as well as high expression of ROS-related genes and enzymes (Wakeel et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCr has no particular biological role in plant cells and is not necessary for plant growth and development (Oliveira \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several plant species have developed different strategies to overcome Cr toxicity. Data in the present study show that THB-81 sequestered up to 552.28 \u0026micro;g g⁻\u0026sup1; DW Cr in roots with no toxicity effect. This is likely due to the vacuolar compartmentalization of Cr or high cell wall binding (Shahandeh and Hossner \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pulford et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shourie et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the contrary, the S-9 and Sindh raya varieties accumulated very low Cr in their roots and shoots, indicating divergent varietal strategies for metal stress response.\u003c/p\u003e\u003cp\u003eThe significant Cr accumulation in stems of Early raya (88.01 \u0026micro;g g⁻\u0026sup1; DW) indicates that Cr is possibly translocated via xylem loading, potentially through non-selective cation channels or sulfur transporters, which are known to facilitate Cr(III) uptake (Prado et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Gill et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; de Oliveira et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, Sindh raya showed minimal Cr uptake (7.83 \u0026micro;g g⁻\u0026sup1; DW in roots), probably due to active efflux, restricted metal influx, and limited translocation to the shoots, which may be due to lignified casparian strips in the root endodermis acting as a barrier to apoplastic movement of Cr. (Yamaji et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) have shown similar results with Zn accumulation in rice. The different varietal responses to Cr exclusion or accumulation are presumably due to the downregulation or upregulation of metal transporters, e.g., HMA4, NRAMP3, or increased chelation of Cr in the rhizosphere caused by high expression of exudate biosynthesis genes rendering Cr in an innocuous form (Clemens \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Shourie et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCr-nutrient interactions\u003c/h2\u003e\u003cp\u003eStrong correlations were observed between Cr and essential metals, such as Fe, Ca, Mg, and K, in the Cr-tolerant varieties (Early raya and THB-81), suggesting competitive metal uptake and a stress response to cope with Cr stress (Vert et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Notably, THB-81 had higher Ca levels in its roots (811.36 \u0026micro;g g⁻\u0026sup1; DW), which could help reduce Cr toxicity by stabilizing membrane integrity and reducing Cr-induced damage (Hirschi \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In contrast, Cr-sensitive varieties (S9 and Sindh raya) exhibited weaker correlations, indicating that they primarily rely on passive exclusion of Cr.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePrincipal component analysis (PCA) and genetic implications\u003c/h2\u003e\u003cp\u003ePrincipal Component Analysis (PCA) was employed to investigate genotype-specific responses to Cr stress and its interaction with other nutrients (Dolezalova et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the present study, PCA distinguished \u003cem\u003eBrassica juncea\u003c/em\u003e varieties based on their physiological responses to Cr stress, categorizing them into several distinct groups. This classification highlights a variety-specific management of Cr stress, with some accumulating Cr and others excluding it. These varietal differences may reflect different strategies in metal transporter expression, detoxification capacity, or intracellular compartmentalization (Shanker et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe close relationship between THB-81 and Early raya in the accumulator group indicates that these varieties may share genetic traits that enhance Cr uptake and sequestration, such as duplicated metal transporter genes or improved root cortical aerenchyma (DalCorso et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Conversely, excluder varieties like S-9 and Sindh raya may carry allelic variations that provide resistance by limiting metal translocation into the xylem. This could occur through thicker exodermal layers or increased activity of FRD3-like efflux transporters (Durrett et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe hypothesis that these varieties use different strategies for chromium uptake, exclusion, and detoxification is supported by the varying spatial distribution of varieties in the PCA analysis. These findings have important practical implications in the phytoremediation of heavy metals. Accumulator varieties show promise for phytoremediation of Cr-contaminated soils, whereas excluder genotypes might be better suited for safe cultivation in polluted areas. This PCA-based classification provides a useful framework for breeding efforts aimed at enhancing Cr tolerance or reducing metal accumulation in edible plant parts.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDistinctive gene expression patterns in\u003c/b\u003e \u003cb\u003eBrassica juncea\u003c/b\u003e \u003cb\u003evarieties under Cr stress\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSindh raya and Early raya showed varied reactions to the gene expression of oxidative-related enzymes when subjected to different levels of Cr. This pronounced varietal response is possibly due to their ability to cope differently with oxidative stress. Exposure of Sindh raya to 1000 \u0026micro;M Cr increased APX (ascorbate peroxidase) expression to 13.74-fold compared to controls. Meanwhile, the levels of FeSOD (Fe-superoxide dismutase) and RBOH (respiratory burst oxidase homolog) remained unchanged despite Cr exposure, suggesting that Sindh raya primarily depends on the APX system for oxidative stress defense. Similar findings were reported by (Jung et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in oilseed rape seedlings exposed to Cd stress. These findings indicate that the two varieties employ different mechanisms to manage ROS generated by metal toxicity (see Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In contrast, Early raya displays a more active and cohesive antioxidant defense system. Unlike Sindh raya, all three genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) showed strong induction, particularly at elevated Cr levels. This synchronized response in Early raya suggests a highly flexible strategy against oxidative stress. FeSOD and RBOH expression are crucial in the plant's response system, which is activated during oxidative stress. For example, (Regon et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that these genes are crucial for aromatic Keteki Joha rice to adapt to oxidative stress caused by ferrous iron (Fe\u0026sup2;⁺). The activation of these ROS-related genes by heavy metals or other abiotic stresses suggests that Early raya has developed a more comprehensive mechanism to handle metal stress (Wang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Małecka et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePractical implications for phytoremediation strategies\u003c/h2\u003e\u003cp\u003eOur data in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e provides a clear, evidence-based framework for selecting the most suitable plant variety to achieve a specific environmental goal, rather than relying on a generic approach (del Real et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Here, Early raya and THB-81 are grouped as Cr accumulators, which tolerate Cr toxicity through Mg accumulation, while S-9 and Sindh raya were shown to be strong excluders of Cr.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe \u003cem\u003eBrassica juncea\u003c/em\u003e varieties are tentatively classified into six groups, considering the influence of other essential elements on Cr uptake and translocation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEarly raya\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrong accumulator\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCo-accumulates Mg, maybe stress-tolerant via internal detoxification\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTHB-81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAccumulator (esp. at high Cr)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePossibly tolerates via moderate Mg accumulation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJS-13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModerate accumulator\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFe uptake seems unrelated to Cr\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS-9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrong excluder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCr-resistant, may avoid uptake or sequester in roots\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSindh raya\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrong excluder\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCr-resistant, similar to S-9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGanj sarhen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeak Cr association, high Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLikely Ca-mediated defense strategy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe different metal strategies studied here can guide future plant breeding and genetic engineering efforts. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, pinpointing specific mechanisms\u0026mdash;such as Ca-mediated defense, Mg-mediated tolerance, or Cr-opposed uptake\u0026mdash;lays a foundation for creating new plant varieties optimized for phytoremediation or food safety. This is a significant and unique finding regarding how different varieties respond to Cr and how it interacts with other essential elements in plants. These varieties could enable safe cultivation in areas contaminated with metal pollutants.\u003c/p\u003e\u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eGiven that Turkey is one of the world\u0026rsquo;s leading chromium exporters and that various regions, particularly regions (i.e., Usak, Manisa, İzmir, Diyarbakir) of Anatolia, suffer from extensive Cr contamination due to leather tanning and mining activities. The findings of this study hold substantial environmental and agricultural significance for safe agriculture and food, not only for Turkey but also for other countries facing similar challenges. This research will be important in addressing global environmental issues. In this context, we explored solutions for alleviating the Cr pollution problem by using suitable plant varieties for remediation or safe cultivation in metal-polluted soils. Our results grouped the \u003cem\u003eBrassica juncea\u003c/em\u003e varieties into three categories: the first group, Cr accumulators (Early raya and THB-81); the second group, intermediate types (JS-13 and Ganj sarhen); and the third group, excluders (S-9 and Sindh raya). We propose that Early raya and THB-81 varieties could be used for phytoremediation of Cr-contaminated sites. In contrast, Sindh raya and S-9 (excluders) could be cultivated for safe agriculture in polluted areas, thereby reducing Cr uptake into the food supply. These variations in Cr uptake, translocation, and accumulation across varieties offer important insights to local authorities, agronomists, and breeders in Turkey and beyond. This helps in developing customized, sustainable approaches to tackle chromium pollution in agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Agricultural Research Institute for providing us with \u003cem\u003eBrassica juncea\u003c/em\u003e L. seeds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Council of Higher Education (YOK)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethrough the Uşak University Research Grant (BAP) under project number 2024/MF002 to Prof. Dr. Abdulrezzak Memon and Prof. Dr. Ahmet Kahraman.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNM performed most of the experiments, while ARM and HA assisted with some of them. NM and ARM wrote the manuscript, with contributions from all authors. NM, ARM, HA, BK, LAB, and AK interpreted\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe data. ARM\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003edesigned and supervised the project. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot applicable\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this article and its supplementary information files. This study ensures complete transparency and accessibility of all data produced or examined.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdnan M, Xiao B, Ali MU, Xiao P, Zhao P, Wang H, Bibi S (2024) Heavy metals pollution from smelting activities: A threat to soil and groundwater. Ecotoxicology and Environmental Safety 274:116189\u003c/li\u003e\n \u003cli\u003eAkcay H, Oguz A, Karapire C (2003) Study of heavy metal pollution and speciation in Buyak Menderes and Gediz river sediments. Water research 37 (4):813-822\u003c/li\u003e\n \u003cli\u003eAkinci IE, Akinci S (2010) Effect of chromium toxicity on germination and early seedling growth in melon (Cucumis melo L.). African Journal of Biotechnology 9 (29):4589-4594\u003c/li\u003e\n \u003cli\u003eAnjum NA, Ahmad I, Pereira ME, Duarte AC, Umar S, Khan NA (2012) The plant family Brassicaceae: contribution towards phytoremediation. 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Frontiers in Plant Science 13:1081624\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":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Brassica juncea, chromium accumulation, gene expression, phytoremediation, sustainable agriculture","lastPublishedDoi":"10.21203/rs.3.rs-7303811/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7303811/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeveral agricultural areas in the Anatolian Plateau of Turkey are contaminated with chromium due to mining and leather tanning activities, posing a serious threat to both safe farming and human health. Cleaning polluted soils for reuse in agriculture is a vital and primary goal of this research. This study aimed to identify suitable Brassica juncea varieties for phytoremediation of metal-contaminated soils in Turkey, with the goal of promoting safe farming practices. Six varieties\u0026mdash;Early Raya, Sindh Raya, S-9, Ganj Sarhen, JS-13, and THB-8\u0026mdash;developed by the Agricultural Institute of Pakistan for drought and salt tolerance, were tested for metal tolerance at different Cr levels (0\u0026ndash;1000 \u0026micro;M). Physiological parameters were recorded, and the contents of Cr, Ca, Fe, Mg, and K in plant tissues were quantified using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Additionally, the gene expression of several enzymes induced by ROS was examined using RT-qPCR. Early raya and Sindh raya under Cr stress showed different expression patterns of these genes, highlighting significant genotypic differences in chromium uptake, nutrient balance, and molecular responses. Our results classified the varieties into three different groups: Early raya and THB-81 as accumulators, Sindh raya, and S-9 as excluders, and Ganj sarhen, and JS13, as intermediate Cr accumulators. This study offers important insights into genotype-specific detoxification strategies. It lays the fundamental groundwork for future breeding programs and phytoremediation research aimed not only at reducing Cr contamination and promoting safe agriculture in contaminated soils in Turkey and other countries facing similar pollution issues.\u003c/p\u003e","manuscriptTitle":"Phytoremediation Potential and Differential Chromium (Cr) Accumulation and Gene Expression in Brassica juncea Varieties for Contaminated Soils in Turkey","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 11:38:10","doi":"10.21203/rs.3.rs-7303811/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-10-14T20:33:44+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-16T06:31:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-16T01:35:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2025-08-14T15:27:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-13T04:30:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2025-08-08T11:42:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3b92b490-1cbd-4b45-95c6-3e94cec89698","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-07T18:39:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-25 11:38:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7303811","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7303811","identity":"rs-7303811","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}