Exogenous hydrogen sulfide increased Nicotiana tabacum L. resistance against drought by the improved photosynthesis and antioxidant system.

Drought stress is an abiotic stressor that impacts photosynthesis, plant growth, and development, leading to decreased crop yields. Sodium hydrosulfide (NaHS), an exogenous additive, has demonstrated potential regulatory effects on plant responses to polyethylene glycol-induced drought stress in tobacco seedlings. Compared to the control, drought stress induced by 15 g/L PEG-6000 significantly reduced several parameters in tobacco seedlings: shoot dry weight (22.83%), net photosynthesis (37.55%), stomatal conductance (33.56%), maximum quantum yield of PSII (Fv/Fm) (11.31%), photochemical quantum yield of PSII (ΦPSII) (25.51%), and photochemical quenching (qP) (18.17%). However, applying NaHS, an H2S donor, mitigated these effects, ultimately enhancing photosynthetic performance in tobacco seedlings. Furthermore, optimal NaHS concentration (0.4 mM) effectively increased leaf stomatal aperture, relative water content (RWC) and root activity, as well as facilitated the absorption of N, K, Mg and S. It also enhanced the accumulation of soluble sugar and proline content to maintain osmotic pressure balance under drought stress. Compared to drought alone, pretreatment with NaHS also bolstered the antioxidant defense system in leaves, leading to 22.93% decrease in hydrogen peroxide (H2O2) content, a 22.19% decrease in malondialdehyde (MDA) content and increased activities of ascorbate peroxidase (APX) by 28.13%, superoxide dismutase (SOD) by 17.07%, peroxidase (POD) by 46.99%, and catalase (CAT) by 65.27%. Consequently, NaHS protected chloroplast structure and attenuated chlorophyll degradation, thus mitigating severe oxidative damage. Moreover, NaHS elevated endogenous H2S levels, influencing abscisic acid (ABA) synthesis and the expression of receptor-related genes, collaboratively participating in the response to drought stress. Overall, our findings provide valuable insights into exogenous NaHS's role in enhancing tobacco drought tolerance. These results lay the foundation for further research utilizing H2S-based treatments to improve crop resilience to water deficit conditions.

Molybdenum inhibited the growth of Phytophthora nicotiana and improved the resistance of Nicotiana tabacum L. against tobacco black shank.

Tobacco black shank (TBS) is a soil-borne fungal disease caused by Phytophthora nicotiana (P. nicotianae), significantly impeding the production of high-quality tobacco. Molybdenum (Mo), a crucial trace element for both plants and animals, plays a vital role in promoting plant growth, enhancing photosynthesis, bolstering antioxidant capacity, and maintaining ultrastructural integrity. However, the positive effect of Mo on plant biotic stress is little understood. This study delves into the inhibitory effects of Mo on P. nicotianae and seeks to unravel the underlying mechanisms. The results showed that 16.32 mg/L of Mo significantly inhibited mycelial growth, altered mycelial morphological structure, damaged mycelial cell membrane, and ultimately led to the leakage of cell inclusions. In addition, 0.6 mg/kg Mo applied in soil significantly reduced the severity of TBS. Mo increased photosynthetic parameters and photosynthetic pigment contents of tobacco leaves, upregulated expression of NtPAL and NtPPO resistance genes, as well as improved activities of SOD, POD, CAT, PPO, and PAL in tobacco plants. Furthermore, Mo could regulate nitrogen metabolism and amino acids metabolism to protect tobacco plants against P. nicotianae infection. These findings not only present an ecologically sound approach to control TBS but also contribute valuable insights to the broader exploration of the role of microelements in plant disease management.

Selenium Improves the Control Efficacy of Phytophthora nicotianae by Damaging the Cell Membrane System and Promoting Plant Energy Metabolism.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, poses a significant threat to tobacco plants. Selenium (Se), recognized as a beneficial trace element for plant growth, exhibited inhibitory effects on P. nicotianae proliferation, disrupting the cell membrane integrity. This action reduced the energy supply and hindered hyphal transport through membrane proteins, ultimately inducing hyphal apoptosis. Application of 8 mg/L Se through leaf spraying resulted in a notable decrease in TBS incidence. Moreover, Se treatment preserved chloroplast structure, elevated chitinase activities, ß-1,3-GA, polyphenol oxidase, phenylalanine ammonia-lyase, and increased hormonal content. Furthermore, Se enhanced flavonoid and sugar alcohol metabolite levels while diminishing amino acid and organic acid content. This shift promoted amino acid degradation and flavonoid synthesis. These findings underscore the potential efficacy of Se in safeguarding tobacco and potentially other plants against P. nicotianae.

Selenium-molybdenum interactions reduce chromium toxicity in Nicotiana tabacum L. by promoting chromium chelation on the cell wall.

Chromium (Cr) is a hazardous heavy metal that negatively affects animals and plants. The micronutrients selenium (Se) and molybdenum (Mo) have been widely shown to alleviate heavy metal toxicity in plants. However, the molecular mechanism of Cr chelation on the cell wall by combined treatment with Se and Mo has not been reported. Therefore, this study aimed to explore the effects of Se-Mo interactions on the subcellular distribution of Cr (50 µM) and on cell wall composition, structure, functional groups and Cr content, in addition to performing a comprehensive analysis of the transcriptome. Our results showed that the cell walls of shoots and roots accumulated 51.0% and 65.0% of the Cr, respectively. Furthermore, pectin in the cell wall bound 69.5%/90.2% of the Cr in the shoots/roots. Se-Mo interactions upregulated the expression levels of related genes encoding galacturonosyltransferase (GAUT), UTP-glucose-1-phosphate uridylyltransferase (UGP), and UDP-glucose-4-epimerase (GALE), involved in polysaccharide biosynthesis, thereby increasing pectin and cellulose levels. Moreover, combined treatment with Se and Mo increased the lignin content and cell wall thickness by upregulating the expression levels of genes encoding cinnamyl alcohol dehydrogenase (CAD), peroxidase (POX) and phenylalanine amino-lyase (PAL), involved in lignin biosynthesis. Fourier-transform infrared (FTIR) spectroscopy results showed that Se + Mo treatment (in combination) increased the number of carboxylic acid groups (-COOH) groups, thereby enhancing the Cr chelation ability. The results not only elucidate the molecular mechanism of action of Se-Mo interactions in mitigating Cr toxicity but also provide new insights for phytoremediation and food safety.

Characterization of a novel nornicotine-degrading strain Mycolicibacterium sp. SMGY-1XX from a nornicotine-degrading consortium and preliminary elucidation of its biodegradation pathway by multi-omics analysis.

Nicotine and nornicotine are all toxic alkaloids involved in the formation of carcinogenic tobacco-specific nitrosamines. Microbes play an important role in removing these toxic alkaloids and their derivatives from tobacco-polluted environments. By now, microbial degradation of nicotine has been well studied. However, limited information is available on the microbial catabolism of nornicotine. In the present study, a nornicotine-degrading consortium was enriched from a river sediment sample and characterized by metagenomic sequencing using a combination of Illumina and Nanopore technologies. The metagenomic sequencing analysis demonstrated that Achromobacter, Azospirillum, Mycolicibacterium, Terrimonas, and Mycobacterium were the dominant genera in the nornicotine-degrading consortium. A total of 7 morphologically distinct bacterial strains were isolated from the nornicotine-degrading consortium. These 7 bacterial strains were characterized by whole genome sequencing and examined for their ability to degrade nornicotine. Based on a combination of 16 S rRNA gene similarity comparisons, 16 S rRNA gene-based phylogenetic analysis, and ANI analysis, the accurate taxonomies of these 7 isolated strains were identified. These 7 strains were identified as Mycolicibacterium sp. strain SMGY-1XX, Shinella yambaruensis strain SMGY-2XX, Sphingobacterium soli strain SMGY-3XX, Runella sp. strain SMGY-4XX, Chitinophagaceae sp. strain SMGY-5XX, Terrimonas sp. strain SMGY-6XX, Achromobacter sp. strain SMGY-8XX. Among these 7 strains, Mycolicibacterium sp. strain SMGY-1XX, which has not been reported previously to have the ability to degrade nornicotine or nicotine, was found to be capable of degrading nornicotine, nicotine as well as myosmine. The degradation intermediates of nornicotine and myosmine by Mycolicibacterium sp. strain SMGY-1XX were determined and the nornicotine degradation pathway in strain SMGY-1XX was proposed. Three novel intermediates, myosmine, pseudooxy-nornicotine, and γ-aminobutyrate, were identified during the nornicotine degradation process. Further, the most likely candidate genes responsible for nornicotine degradation in Mycolicibacterium sp. strain SMGY-1XX were identified by integrating genomic analysis, transcriptomic analysis, and proteomic analysis. The findings in this study will help to expand our understanding on the microbial catabolism of nornicotine and nicotine and provide new insights into the nornicotine degradation mechanism by consortia and pure culture, laying a foundation for the application of strain SMGY-1XX for the removal, biotransformation, or detoxification of nornicotine.

Selenium in soil-plant system: Transport, detoxification and bioremediation.

Selenium (Se) is an essential micronutrient for humans and a beneficial element for plants. However, high Se doses always exhibit hazardous effects. Recently, Se toxicity in plant-soil system has received increasing attention. This review will summarize (1) Se concentration in soils and its sources, (2) Se bioavailability in soils and influencing factors, (3) mechanisms on Se uptake and translocation in plants, (4) toxicity and detoxification of Se in plants and (5) strategies to remediate Se pollution. High Se concentration mainly results from wastewater discharge and industrial waste dumping. Selenate (Se [VI]) and selenite (Se [IV]) are the two primary forms absorbed by plants. Soil conditions such as pH, redox potential, organic matter and microorganisms will influence Se bioavailability. In plants, excessive Se will interfere with element uptake, depress photosynthetic pigment biosynthesis, generate oxidative damages and cause genotoxicity. Plants employ a series of strategies to detoxify Se, such as activating antioxidant defense systems and sequestrating excessive Se in the vacuole. In order to alleviate Se toxicity to plants, some strategies can be applied, including phytoremediation, OM remediation, microbial remediation, adsorption technique, chemical reduction technology and exogenous substances (such as Methyl jasmonate, Nitric oxide and Melatonin). This review is expected to expand the knowledge of Se toxicity/detoxicity in soil-plant system and offer valuable insights into soils Se pollution remediation strategies.

Selenium and molybdenum synergistically alleviate chromium toxicity by modulating Cr uptake and subcellular distribution in Nicotiana tabacum L.

Chromium (Cr) is a harmful heavy metal that poses a serious threat to plants and animals. Selenium (Se) and molybdenum (Mo) are two beneficial elements for plant growth and resistance. However, their interactive effects on Cr uptake and distribution are poorly understood. Therefore, a hydroponics experiment was conducted to explore the effects of the use of Se and Mo alone and simultaneously on mitigating Cr toxicity. In this study, Nicotiana tabacum L. seedlings were exposed to control, 50 µM Cr, 50 µM Cr + 2 µM Se, 50 µM Cr + 1 µM Mo, or 50 µM Cr + 2 µM Se + 1 µM Mo in Hoagland solution. After 2 weeks, the plant biomass, Cr, Se and Mo contents, photosynthesis, leaf ultrastructure, antioxidant system, subcellular distribution and associated gene expression in Nicotiana tabacum L. were determined. The results showed that simultaneous use of Se and Mo promoted tobacco growth under Cr stress, as evidenced by reducing reactive oxygen species (ROS) content and reducing Cr translocation factor (TF) and inducing a 51.3% reduction in Cr content in shoots. Additionally, Se-Mo interactions increased the levels of glutathione (GSH) and phytochelatin (PC) and the distribution of Cr in the cell walls and organelles. Furthermore, the relative expression of PCS1 was upregulated, while those of NtST1 and MSN1 were downregulated. The results concluded that the simultaneous use of Se and Mo effectively alleviated Cr toxicity in Nicotiana tabacum L., which not only offers an efficient way for crops to resist Cr toxicity but also provides evidence for the benefit of Se combined with Mo.

Evaluation of the cadmium phytoextraction potential of tobacco (Nicotiana tabacum) and rhizosphere micro-characteristics under different cadmium levels.

In this study, a field-scale and pot experiment were performed to evaluate the remedial efficiency of Cd contaminated soil by tobacco and explore rhizosphere micro-characteristics under different cadmium levels, respectively. The results indicated that tobacco could remove 12.9 % of Cd from soil within a short growing period of 80 d. The pot experiment revealed that tobacco could tolerate soil Cd concentrations up to 5.8 mg kg-1 and bioaccumulate 68.1 and 40.8 mg kg-1 Cd in shoots and roots, respectively. The high Cd bioaccumulation in tobacco might be attributed to strong acidification in the rhizosphere soil and the increase in Cd bioavailability. Rhizobacteria did not appear to be involved in Cd mobilization. In contrast, tobacco tended to enrich sulfate-reducing bacteria (such as Desulfarculaceae) under high Cd treatment (5.8 mg kg-1) but enrich plant growth-promoting bacteria (such as Bacillus, Dyadobacter, Virgibacillus and Lysobacter) to improve growth under low Cd treatment (0.2 mg kg-1), suggesting that tobacco employed different microbes for responding to Cd stress. Our results demonstrate the advantages of using tobacco for bioremediating Cd contaminated soil and clarify the rhizosphere mechanisms underlying Cd mobilization and tolerance.

Comparison of selenite and selenate in alleviation of drought stress in Nicotiana tabacum L.

Exogenous selenium (Se) improves the tolerance of plants to abiotic stress. However, the effects and mechanisms of different Se species on drought stress alleviation are poorly understood. This study aims to evaluate and compare the different effects and mechanisms of sodium selenate (Na2SeO4) and sodium selenite (Na2SeO3) on the growth, photosynthesis, antioxidant system, osmotic substances and stress-responsive gene expression of Nicotiana tabacum L. under drought stress. The results revealed that drought stress could significantly inhibit growth, whereas both Na2SeO4 and Na2SeO3 could significantly facilitate the growth of N. tabacum under drought stress. However, compared to Na2SeO3, Se application as Na2SeO4 induced a significant increase in the root tip number and number of bifurcations under drought stress. Furthermore, both Na2SeO4 and Na2SeO3 displayed higher levels of photosynthetic pigments, better photosynthesis, and higher concentrations of osmotic substances, antioxidant enzymes, and stress-responsive gene (NtCDPK2, NtP5CS, NtAREB and NtLEA5) expression than drought stress alone. However, the application of Na2SeO4 showed higher expression levels of the NtP5CS and NtAREB genes than Na2SeO3. Both Na2SeO4 and Na2SeO3 alleviated many of the deleterious effects of drought in leaves, which was achieved by reducing stress-induced lipid peroxidation (MDA) and H2O2 content by enhancing the activity of antioxidant enzymes, while Na2SeO4 application showed lower H2O2 and MDA content than Na2SeO3 application. Overall, the results confirm the positive effects of Se application, especially Na2SeO4 application, which is markedly superior to Na2SeO3 in the role of resistance towards abiotic stress in N. tabacum.

[Advances in plant heavy metal transporter P1B-ATPases].

P1B-ATPases are a group of proteins that can transport heavy metal ions across membranes by hydrolyzing ATP and they are a subclass of the P-type ATPase family. It was found that P1B-ATPases are mainly responsible for the active transport of heavy metal ions in plants and play an important role in the regulation of heavy metal homeostasis in plants. In this paper, we dissusses the mechanism of P1B-ATPases from the structure and classification of P1B-ATPases, and review the current research progress in the function of P1B-ATPases, in order to provide reference for future research and application of P1B-ATPases in improving crop quality and ecological environment management.

Differential cadmium translocation and accumulation between Nicotiana tabacum L. and Nicotiana rustica L. by transcriptome combined with chemical form analyses.

Cadmium (Cd) is a severely toxic and carcinogenic heavy metal. Cigarette smoking is one of the major source of Cd exposure in humans. Nicotiana tabacum is primarily a leaf Cd accumulator, while Nicotiana rustica is a root Cd accumulator among Nicotiana species. However, little is known about the mechanisms of differential Cd translocation and accumulation in Nicotiana. To find the key factors, Cd concentration, Cd chemical forms, and transcriptome analysis were comparatively studied between N. tabacum and N. rustica under control or 10 µM Cd stress. The leaf/root Cd concentration ratio of N. tabacum was 2.26 and that of N. rustica was 0.14. The Cd concentration in xylem sap of N. tabacum was significantly higher than that of N. rustica. The root of N. tabacum had obviously higher proportion of ethanol extractable Cd (40%) and water extractable Cd (16%) than those of N. rustica (16% and 6%). Meanwhile the proportion of sodium chloride extracted Cd in N. rustica (71%) was significantly higher than that in N. tabacum (30%). A total of 30710 genes expressed differentially between the two species at control, while this value was 30,294 under Cd stress, among which 27,018 were collective genes, manifesting the two species existed enormous genetic differences. KEGG pathway analysis showed the phenylpropanoid biosynthesis pathway was overrepresented between the two species under Cd stress. Several genes associated with pectin methylesterase, suberin and lignin synthesis, and heavy metal transport were discovered to be differential expressed genes between two species. The results suggested that the higher accumulation of Cd in the leaf of N. tabacum depends on a comprehensive coordination of Cd transport, including less cell wall binding, weaker impediment by the Casparian strip, and efficient xylem loading.

Combination of ß-Aminobutyric Acid and Ca2+ Alleviates Chilling Stress in Tobacco (Nicotiana tabacum L.).

Chilling is a major abiotic factor limiting the growth, development, and productivity of plants. ß-aminobutyric acid (BABA), a new environmentally friendly agent, is widely used to induce plant resistance to biotic and abiotic stress. Calcium, as a signaling substance, participates in various physiological activities in cells and plays a positive role in plant defense against cold conditions. In this study, we used tobacco as a model plant to determine whether BABA could alleviate chilling stress and further to explore the relationship between BABA and Ca2+. The results showed that 0.2 mM BABA significantly reduced the damage to tobacco seedlings from chilling stress, as evidenced by an increase in photosynthetic pigments, the maintenance of cell structure, and upregulated expression of NtLDC1, NtERD10B, and NtERD10D. Furthermore, 0.2 mM BABA combined with 10 mM Ca2+ increased the fresh and dry weights of both roots and shoots markedly. Compared to that with single BABA treatment, adding Ca2+ reduced cold injury to the plant cell membrane, decreased ROS production, and increased antioxidant enzyme activities and antioxidant contents. The combination of BABA and Ca2+ also improved abscisic acid and auxin contents in tobacco seedlings under chilling stress, whereas ethylene glycol-bis (ß-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) reversed the effects of BABA. These findings suggested that BABA enhances the cold tolerance of tobacco and is closely related to the state of Ca2+ signaling.

Differential expression of vacuolar and defective cell wall invertase genes in roots and seeds of metalliferous and non-metalliferous populations of Rumex dentatus under copper stress.

Acid invertase activities in roots and young seeds of a metalliferous population (MP) of Rumex dentatus were previously observed to be significantly higher than those of a non-metalliferous population (NMP) under Cu stress. To date, no acid invertase gene has been cloned from R. dentatus. Here, we isolated four full-length cDNAs from the two populations of R. dentatus, presumably encoding cell wall (RdnCIN1 and RdmCIN1 from the NMP and MP, respectively) and vacuolar invertases (RdnVIN1 and RdmVIN1 from the NMP and MP, respectively). Unexpectedly, RdnCIN1 and RdmCIN1 most likely encode special defective invertases with highly attenuated sucrose-hydrolyzing capacity. The transcript levels of RdmCIN1 were significantly higher than those of RdnCIN1 in roots and young seeds under Cu stress, whereas under control conditions, the former was initially lower than the latter. Unexpected high correlations were observed between the transcript levels of RdnCIN1 and RdmCIN1 and the activity of cell wall invertase, even though RdnCIN1 and RdmCIN1 do not encode catalytically active invertases. Similarly, the transcript levels of RdmVIN1 in roots and young seeds were increased under Cu stress, whereas those of RdnVIN1 were decreased. The high correlations between the transcript levels of RdnVIN1 and RdmVIN1 and the activity of vacuolar invertase indicate that RdnVIN1 and RdmVIN1 might control distinct vacuolar invertase activities in the two populations. Moreover, a possible indirect role for acid invertases in Cu tolerance, mediated by generating a range of sugars used as nutrients and signaling molecules, is discussed.

[Spatial distribution of sulfur dioxide around a tobacco bulk-curing workshop cluster].

In order to manifest lower energy consumption and less labor employment, and provide the theoretical basis for constructing environmentally friendly modem tobacco agriculture, this paper analyzed gas composition of the chimney from a bulk-curing barn and the dispersion of sulfur dioxide (SO2) around the workshop cluster using ecom-J2KN flue gas analyzer and air sampler. During curing, the concentrations of carbon dioxide (CO2) and SO2 in the chimney were both highest at 38 degrees C, while the concentration of nitrogen oxides (NOx) was highest at 42 degrees C. The emission concentration of SO2 from the chimney was 1327.60-2218.40 mg x m(-3). Average SO2 emission would decrease by 49.7% through adding 4.0% of a sulfur-fixed agent. The highest concentrations of SO2 in the surface soil appeared at the yellowing stage. SO2 concentration in horizontal direction localized at 43-80 m exceeded 0.5 mg x m(-3). The highest concentration of SO2 (0.57 mg x m(-3)) was observed at 50 m. At 50 m in the downstream wind direction of the workshop cluster, SO2 concentration in vertical direction localized at 0.9-1.8 m exceeded 0.5 mg x m(-3), and the highest concentration of SO2 in vertical direction was 0.65 mg x m(-3) at 1.6 m. During curing, the average concentration of SO2 was decreased by 0.43 mg x m(-3) by using the sulfur-fixed agent. The polluted boundary was localized at 120 m in the downstream wind direction of the workshop cluster.

Comparative study of root growth and sucrose-cleaving enzymes in metallicolous and non-metallicolous populations of Rumex dentatus under copper stress.

Sucrose metabolism in roots of metallophytes is very important for root growth and maintenance of heavy metal tolerance. However, rare researches have been carried out on this topic so far. We tested here a hypothesis that roots of copper-tolerant plants should manifest higher activities of sucrose-cleaving enzymes than non-tolerant plants for maintaining root growth under Cu stress. Plants of two contrasting populations of metallophyte Rumex dentatus, one from an ancient Cu mine (MP) and the other from a non-mine site (NMP), were treated with Cu in controlled experiments. Cu treatment resulted in a higher root biomass and root/shoot biomass ratio in MP compared to NMP. More complicated root system architecture was showed in MP under Cu stress. Activities and transcript levels of acid invertase as well as contents of sucrose and reducing sugar in MP were elevated under Cu treatment, while activities of neutral/alkaline invertase and sucrose synthase showed no significant differences between two populations. The results indicate important roles of acid invertase in governing root growth under Cu stress.

Differential expression of acid invertase genes in roots of metallicolous and non-metallicolous populations of Rumex japonicus under copper stress.

Recent evidence indicates that during copper (Cu) stress, the roots of metallicolous plants manifest a higher activity of acid invertase enzymes, which are rate-limiting in sucrose catabolism, than non-metallicolous plants. To test whether the higher activity of acid invertases is the result of higher expression of acid invertase genes, we isolated partial cDNAs for acid invertases from two populations of Rumex japonicus (from metalliferous and non-metalliferous soils), determined their nucleotide sequences, and designed primers to measure changes in transcript levels during Cu stress. We also determined the growth of the plants' roots, Cu accumulation, and acid invertase activities. The seedlings of R. japonicus were exposed to control or 20 µM Cu(2+) for 6d under hydroponic conditions. The transcript level and enzyme activity of acid invertases in metallicolous plants were both significantly higher than those in non-metallicolous plants when treated with 20 µM. Under Cu stress, the root length and root biomass of metallicolous plants were also significantly higher than those of non-metallicolous plants. The results suggested that under Cu stress, the expression of acid invertase genes in metallicolous plants of R. japonicus differed from those in non-metallicolous plants. Furthermore, the higher acid invertase activities of metallicolous plants under Cu stress could be due in part to elevated expression of acid invertase genes.

Effects of copper on phenology and reproduction in Rumex dentatus from metalliferous and non-metalliferous sites.

The responses of phenology and reproductive traits to copper stress in two populations of Rumex dentatus were comparatively studied with pot culture experiments. Seeds used for the experiments were, respectively, collected from metalliferous and normal soils. It was found that the responses of phenology and reproductive traits to Cu treatment between the two populations were significantly different. Compared to the non-metallicolous population, the metallicolous population of R. dentatus had a short life cycle, large reproductive effort, and high fertility under Cu stress. In addition, the reproductive effort in metallicolous population of R. dentatus was maintained at the expense of a curtailment of vegetative development. The results suggested that change in phenological traits and more resources allocation to reproduction might play an important role in the adaptation of metallicolous population of R. dentatus to the Cu-enriched mine soils.