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Additional file 1. Transmission electron microscope image of the silver nanoparticles used in the study. Nanoparticles were obtained in solid form from US Research Nanomaterials, Inc. (Houston, TX, USA), and were applied to soil in this form.

Phytoextraction makes use of trace element-accumulating plants that concentrate the pollutants in their tissues. Pollutants can be then removed by harvesting plants. The success of phytoextraction depends on trace element availability to the roots and the ability of the plant to intercept, take up, and accumulate trace elements in shoots. Current phytoextraction practises either employ hyperaccumulators or fast-growing high biomass plants; the phytoextraction process may be enhanced by soil amendments that increase trace element availability in the soil. This review will focus on the role of plant-associated bacteria to enhance trace element availability in the rhizosphere. We report on the kind of bacteria typically found in association with trace element – tolerating or – accumulating plants and discuss how they can contribute to improve trace element uptake by plants and thus the efficiency and rate of phytoextraction. This enhanced trace element uptake can be attributed to a microbial modification of the absorptive properties of the roots such as increasing the root length and surface area and numbers of root hairs, or by increasing the plant availability of trace elements in the rhizosphere and the subsequent translocation to shoots via beneficial effects on plant growth, trace element complexation and alleviation of phytotoxicity. An analysis of data from literature shows that effects of bacterial inoculation on phytoextraction efficiency are currently inconsistent. Some key processes in plant–bacteria interactions and colonization by inoculated strains still need to be unravelled more in detail to allow full-scale application of bacteria assisted phytoremediation of trace element contaminated soils.

Lipid peroxidation in leaves and roots of A. thaliana wild-type and spl7 knockout plants. Nineteen-days-old plants were exposed for 24 h to 2 μM CuSO4, 5 μM CdSO4 or grown under control conditions. Data are mean ± S.E. of 4 biological replicates. Significant treatment differences (P

Forward and reverse primers used to determine gene expression levels via quantitative real-time PCR. E-E-jn, exon-exon junction; E-I-b, exon-intron boundary; YLS, yellow-leaf-specific; UBQ10, ubiquitin; ACT, actin ; EF, elongation factor; APS, ATP sulfurylase; SULTR, sulfate transporter; LAC, laccase; CSD, Cu/Zn superoxide dismutase; FSD, Fe superoxide dismutase. *Pri-miRNA primer concentrations were increased to 900 nM. (DOCX 18 kb)

Verification of the T-DNA insertion in the spl7 mutant. Primers were designed using T-DNA primer design. (DOCX 32 kb)

Quantitative real-time PCR parameters according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines derived from Bustin et al. [51]. (DOCX 16 kb)

Additional file 1. Soil characteristics analyzed in 2012 and 2013. a organic matter, b electrical conductivity of a saturated soil paste extract (ECe), c Total soil N by Kjeldahl method.

Additional file 2. VAP test - Comparison of the root system architectures. Vertical Agar Plate test. Comparison of the root system architectures of non-inoculated and inoculated Arabidopsis seedlings through the 2D pictures created with the RootNav image analysis tool (University of Nottingham).

Cadmium (Cd) and zinc (Zn) were applied to durum wheat seedlings on hydroponics in concentrations of 50 μM and 600 μM respectively, causing about 50% relative growth inhibition (RGI) at the end of a 10-day-exposure period. Both metals provoked visual toxicity symptoms such as root browning and leaf chlorosis in Cd-exposed seedlings and root colour lightening and leaf necrotic spots in Zn-exposed seedlings. On a biochemical level, the apparent phytotoxicity was accompanied by oxidative stress-related responses such as increased lipid peroxidation and electrolyte leakage as well as increased activities of superoxide dismutase and catalase.

We explored the seed-associated bacterial endophytic microbiome in seeds of the endemic holoparasitic species Cistanche armena from a saline and arid habitat in Armenia. A combination of culture-dependent and molecular techniques was employed for identifying the seed endomicrobiome (culturable and unculturable). From surface-sterilized seeds, 10 phyla, comprising 256 endophytic bacterial genera, were identified. Of the culturable strains, we also investigated the plant growth-promoting (PGP) traits. Most of the isolates were spore forming, halotolerant and alkaliphile Bacillus spp., indicating that the endophytic bacteria of C. armena seeds own traits related to the natural habitat of their host plant. Our results confirm that Bacillus species are common and dominated endophytes from plants growing on saline and arid soils. Pantoea spp. and Stenotrophomonas spp. are more favourable PGP endophytes in seeds of C. armena. The PGP traits of these bacteria, such as production of indole, a precursor of auxin, ACC-deaminase and organic acids have the potential to improve the tolerance of their host plants against the abiotic stresses present in their natural habitat. To the best of our knowledge, this is the first report concerning bacterial seed endophytes of the C. armena. The manuscript was prepared under ‘Partnership agreement governing the joint supervision and awarding of a doctorate diploma between Jan Kochanowski University in Kielce (Poland) and Hasselt University (Belgium)’ (K.P.). We thank Dr. Yuliya Krasylenko for taking photographs under a zoom microscope. The author acknowledges financial support through the project ‘Development Accelerator of the Jan Kochanowski University of Kielce’, co-financed by the European Union under the European Social Fund (K.P., POWR.03.05.00-00-Z212/18, 2019– 2023). This study was supported by grants from the Jan Kochanowski University (K.R., 666 065, 2019) and (W.K., K.P., SUPB.RN. 21.235, 2021-2022). The field research in this study in Armenia was partially financed by the National Geographic grant (R.P., GEFNE 192-16, 2017). This study was also supported by a BOF-BILA grant from Hasselt University Belgium BOF21BL12 (K.P., J.V., 2021–2022) and the Hasselt University Methusalem project (J.V., 08M03VGRJ).

BACKGROUND: The gut microbiome plays an essential role in human health. Despite the link between air pollution exposure and various diseases, its association with the gut microbiome during susceptible life periods remains scarce. OBJECTIVES: In this study, we examined the association between black carbon particles quantified in prenatal and postnatal biological matrices and bacterial richness and diversity measures, and bacterial families. METHODS: A total of 85 stool samples were collected from 4- to 6-y-old children enrolled in the ENVIRonmental influence ON early AGEing birth cohort. We performed 16S rRNA gene sequencing to calculate bacterial richness and diversity indices (Chao1 richness, Shannon diversity, and Simpson diversity) and the relative abundance of bacterial families. Black carbon particles were quantified via white light generation under femtosecond pulsed laser illumination in placental tissue and cord blood, employed as prenatal exposure biomarkers, and in urine, used as a post-natal exposure biomarker. We used robust multivariable-adjusted linear models to examine the associations between quantified black carbon loads and measures of richness (Chao1 index) and diversity (Shannon and Simpson indices), adjusting for parity, season of delivery, sequencing batch, age, sex, weight and height of the child, and maternal education. Additionally, we performed a differential relative abundance analysis of bacterial families with a correction for sampling fraction bias. Results are expressed as percentage difference for a doubling in black carbon loads with 95% confidence interval (CI). RESULTS: Two diversity indices were negatively associated with placental black carbon [Shannon: -4.38% (95% CI: -8.31%, -0.28%); Simpson: -0.90% (95% CI: -1.76%, -0.04%)], cord blood black carbon [Shannon: -3.38% (95% CI: -5.66%, -0.84%); Simpson: -0.91 (95% CI: -1.66%, -0.16%)], and urinary black carbon [Shannon: -3.39% (95% CI: -5.77%, -0.94%); Simpson: -0.89% (95% CI: -1.37%, -0.40%)]. The explained variance of black carbon on the above indices varied from 6.1% to 16.6%. No statistically significant associations were found between black carbon load and the Chao1 richness index. After multiple testing correction, placental black carbon was negatively associated with relative abundance of the bacterial families Defluviitaleaceae and Marinifilaceae, and urinary black carbon with Christensenellaceae and Coriobacteriaceae; associations with cord blood black carbon were not statistically significant after correction. CONCLUSION: Black carbon particles quantified in prenatal and postnatal biological matrices were associated with the composition and diversity of the childhood intestinal microbiome. These findings address the influential role of exposure to air pollution during pregnancy and early life in human health. https://doi.org/10.1289/EHP11257. ispartof: Environ Health Perspect vol:131 issue:1 pages:17010- ispartof: location:United States status: published

The inclusion of bioaugmented low-cost biochar in current wastewater treatment technologies is a promising way to enhance the removal and degradation of emerging contaminants. In this paper, the properties of two wood waste biochars (wood waste mix - AB, and date palm fiber wood - PDF), and coffee bean husks (COF), produced at four temperatures (350, 450, 500, 550°C) were compared, and investigated in the presence of Geobacter sulfurreducens or a mixed freshwater stream bacterial culture to understand their potential for the adsorption and biotransformation of two types of pesticides (thiacloprid, pirimicarb), and two pharmaceuticals (ibuprofen, diclofenac). Biochar yield was similar for all three biochars and ranged between 30 and 35%. The ash content of PDF and COF was significantly higher than AB. pH and electrical conductivity (EC) were initially high for COF (pH: 7.4–8; EC: 3–4.27 mS/cm) and PDF (pH: 7.7–10.1; EC: 4–6.24 mS/cm) after 24 h, but stabilized at neutral pH and − (550°C), and 846 mg/L sulphate (350°C). Lower pyrolysis temperatures reduced leachable anions. The biochars were highly (ultra)microporous with little meso- and macroporosity. The adsorption experiments showed that AB and COF biochars were both suited to sorb more than 90% of the initially spiked 10 ppm pirimicarb, AB removed 50.2% of the initial diclofenac concentration compared to only 5% for the no-biochar control, and both biochars could remove about 55% of the initially spiked thiacloprid, and 40% of the ibuprofen. In the presence of a mixed culture, on average 30% more thiacloprid and ibuprofen was removed from the supernatant by AB and COF than the sterile control. This work shows that selected wood-waste feedstocks and low pyrolysis temperature can produce environmentally-safe biochars that have suitable characteristics to sorb emergent pollutants from water. These materials could be further studied in multi-pollution sorption/competition experiments, and in larger environmental wastewater treatment systems.

The integration of phytoremediation and biostimulation can improve pollutant removal from the environment. Plant secondary metabolites (PSMs), which are structurally related to xenobiotics, can stimulate the presence of microbial community members, exhibiting specialized functions toward detoxifying, and thus mitigating soil toxicity. In this study, we evaluated the effects of enrichment of 4-chloro-2-methylphenoxyacetic acid (MCPA) contaminated soil (unplanted and zucchini-planted) with syringic acid (SA) on the bacterial community structure in soil, the rhizosphere, and zucchini endosphere. Additionally, we measured the concentration of MCPA in soil and fresh biomass of zucchini. The diversity of bacterial communities differed significantly between the studied compartments (i.e., unplanted soil, rhizospheric soil, and plant endosphere: roots or leaves) and between used treatments (MCPA or/and SA application). The highest diversity indices were observed for unplanted soil and rhizosphere. Although the lowest diversity was observed among leaf endophytes, this community was significantly affected by MCPA or SA: the compounds applied separately favored the growth of Actinobacteria (especially Pseudarthrobacter), while their simultaneous addition promoted the growth of Firmicutes (especially Psychrobacillus). The application of MCPA + SA together lead also to enhanced growth of Pseudomonas, Burkholderia, Sphingomonas, and Pandoraea in the rhizosphere, while SA increased the occurrence of Pseudomonas in leaves. In addition, SA appeared to have a positive influence on the degradative potential of the bacterial communities against MCPA: its addition, followed by zucchini planting, significantly increased the removal of the herbicide (50%) from the soil without affecting, neither positively nor negatively, the plant growth. The Council of the National Science Centre in Poland ETIUDA 7–funded the project “Cucurbits and their plant secondary metabolites as stimulators of biological soil remediation contaminated with phenoxy herbicides” [No. 2019/32/T/NZ9/00403]. This work was also supported by the UHasselt Methusalem project 08M03VGRJ.

This field study aimed to assess the baseline conditions of a long-term shooting range in Argentina polluted with 428 mg kg(-1) lead (Pb) to evaluate the establishment and development of Helianthus petiolaris plants and address the efficacy of the phytomanagement strategy through: (i) element accumulation in plant tissues; (ii) rhizosphere bacterial diversity changes by Illumina Miseq (TM), and (iii) floral water and essential oil yield, composition, and element concentration by GC-MS and ICP. After one life cycle growing in the polluted sites, in the roots of Helianthus petiolaris plants, Pb concentration was between 195 and 304 mg kg(-1) Pb. Only a limited fraction of the Pb was translocated to the aerial parts. The predominance of the genus Serratia in the rhizosphere of Helianthus petiolaris plants cultivated in the polluted sites and the decrease in the essential oil yield were some effects significantly associated with soil Pb concentration. No detectable Pb concentration was found in the floral water and essential oil obtained. Extractable Pb concentration in the soil reduced between 28% and 45% after the harvest. This work was supported by the Ibero-American Development Bank under grant FONTAGRO (ATN/RF-16110-RG), Pluriannuals Projects of Investigation-CONICET (PIP0730), and the Centre for Environmental Sciences of Hasselt University, Belgium, under grant BOF-BILA No. 8546. Thanks to Andrea R. Costantino and Natalia Lazzarini from the Instituto Química del Sur, Bahía Blanca, Bs As, Argentina, for their collaboration on the GC–MS analysis of the essential oils.

Cable bacteria are an emerging class of electroactive organisms that sustain unprecedented long-range electron transport across centimeter-scale distances. The local pathways of the electrical currents in these filamentous microorganisms remain unresolved. Here, the electrical circuitry in a single cable bacterium is visualized with nanoscopic resolution using conductive atomic force microscopy. Combined with perturbation experiments, it is demonstrated that electrical currents are conveyed through a parallel network of conductive fibers embedded in the cell envelope, which are electrically interconnected between adjacent cells. This structural organization provides a fail-safe electrical network for long-distance electron transport in these filamentous microorganisms. The observed electrical circuit architecture is unique in biology and can inspire future technological applications in bioelectronics. R.T.E. and R.B. contributed equally to this work. The authors thank the colleagues from X-LAB from Hasselt University and the Microbial Electricity team from University of Antwerp for discussions and feedback. Special thanks to H. T. S. Boschker, I. Cardinaletti, J. Drijkoningen, J. L. Hou, and S. Thijs for their insights and discussions. Thanks to K. Ceyssens and T. Custers for the graphics. This research was financially supported by the Research Foundation Flanders (FWO project grant G031416N to FJRM and JM, FWO aspirant grant 1180517N to RB) and Dutch Research Council (NWO Vici grant 016.VICI.170.072 to FJRM). All measurements and data analysis were performed by R.T.E. and R.B. in equal contribution. J.M. and B.C. coordinated the study. Conceptualization and discussion were done by J.M., R.V., B.C., R.C., R.T.E., and R.B. Cable bacteria enrichment and fiber sheath extraction was performed by S.H.-M., R.B., and F.J.R.M. Funding was acquired by J.M., J.V., and F.J.R.M. Writing was done by R.B., R.T.E., J.M., B.C., and F.J.R.M., with contributions from all authors.

The commonly used Arabidopsis thaliana natural accessions Columbia (Col-0) and Wassilewskija (Ws) are known to differ in their metal sensitivity, with Col-0 being more sensitive to copper (Cu) and cadmium (Cd) than Ws. As both Cu and Cd are known to affect Cu homeostasis, it was investigated whether this process is part of an accession-specific mechanism underlying their difference in metal sensitivity. As roots are the first contact point during metal exposure, responses were compared between roots of both accessions of hydroponically grown plants exposed to excess Cu or Cd for 24 and 72 h.Root Cu levels increased in both accessions under Cu and Cd exposure. However, under Cu exposure, the downregulation of Cu transporter (COPT) genes in combination with a more pronounced upregulation of metallothionein gene MT2b indicated that Ws plants coped better with the elevated Cu concentrations. The Cdinduced disturbance in Cu homeostasis was more efficiently counteracted in roots of Ws plants than in Col0 plants. This was indicated by a higher upregulation of the SPL7-mediated pathway, crucial in the regulation of the Cu homeostasis response.In conclusion, maintaining the Cu homeostasis response in roots is key to accession-specific differences in Cu and Cd sensitivity. This work was supported by BOF funding from Hasselt University through a PhD grant for Rafaela Amaral dos Reis [grant number BOF12NI28]. Additional funding came from FWO projects [grant numbers G0C7518 N, G0B6716 N].

Poplar clones were studied for their phytoextraction capacity in the second growth cycle (6-year growth) on a site in the Belgian Campine region, which is contaminated with Cd and Zn via historic atmospheric deposition of nearby zinc smelter activities. The field trial revealed regrowth problems for some clones that could not be predicted in the first growth cycle. Four allometric relations were assessed for their capacity to predict biomass yield in the second growth cycle. A power function based on the shoot diameter best estimates the biomass production of poplar with R-2 values between 0.94 and 0.98. The woody biomass yield ranged from 2.1 to 4.8 ton woody Dry Mass (DM) ha(-1) y(-1). The primary goal was to reduce soil concentrations of metals caused by phytoextraction. Nevertheless, increased metal concentrations were determined in the topsoil. This increase can partially be explained by the input of metals from deeper soil layers in the top soil through litterfall. The phytoextraction option with poplar short rotation coppice in this setup did not lead to the intended soil remediation in a reasonable time span. Therefore, harvest of the leaf biomass is put forward as a crucial part of the strategy for soil remediation through Cd/Zn phytoextraction. The research project was financially supported by the European Commission within the framework of the Interreg IVb project "Accelerating Renewable Energies through valorisation of Biogenic Organic Raw Material" (ARBOR).

When terrestrial environments get contaminated with long-lived gamma emitting radionuclides, plants that grow in these contaminated areas are exposed to gamma radiation during consecutive generations. Therefore it is important to evaluate the gamma induced stress response in plants in and between generations. The objective of this research is to reveal differences at the level of the antioxidative stress response between generations with a different radiation history. An experiment was conducted in which 7-days old Arabidopsis thaliana plants were exposed for 14 days to four different gamma dose rates: 22 mGy/h, 38 mGy/h, 86 mGy/h and 457 mGy/h. Two different plant groups were used: plants that were not exposed to gamma radiation before (P0) and plants that received the aforementioned gamma treatment during their previous generation (S1). Growth, the concentration of the antioxidants ascorbate and glutathione, a number of antioxidative enzyme activities and their gene transcript levels were analysed. A dose-rate dependent induction was seen for catalase (CAT) and guaiacol peroxidase (GPX) in the roots and for syringaldazine peroxidase (SPX) in the shoots. Differences between the two generations were observed for CAT and GPX in the roots, where a significantly higher activity of these reactive oxygen species (ROS) detoxifying enzymes was observed in the S1 generation. For SPX in the shoots, a dose dependent upregulation was observed in the PO generation. However, high SPX activities were present for all doses in the S1 generation. These differences in enzyme activity between generations for SPX and GPX and the involvement of these enzymes in cell wall biosynthesis, suggest an important role for cell wall strengthening in the response to gamma irradiation. (C) 2016 Elsevier Ltd. All rights reserved. This project was supported by the European project COMET (7th PCRD EURATOM Contract Number: Fission-2012-3.4.1-604794) (www.comet-radioecology.org) and the Belgian nuclear research centre (SCK CEN).

The reactive oxygen species (ROS)-signaling pathway is very important in heavy metal toxicity. Induction of the antioxidative defense mechanism, comprising ROS-scavenging enzymes and metabolites, in plants after environmental uranium contamination has been insufficiently studied in the past. This study aimed to analyze oxidative stress related responses in Arabidopsis thaliana after uranium exposure. Seventeen-day-old seedlings were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 3 days. After exposure to 100 μM uranium, a decrease in fresh weight for leaves and roots was observed, leaves colored anthocyanous and roots were stunted and yellow. To reveal the importance of oxidative stress in uranium toxicity, alterations in ROS-scavenging enzymes were studied at protein and transcriptional level. Superoxide dismutase (SOD) capacities increased in leaves and roots after exposure to 100 μM uranium but no differences were observed for catalase (CAT) capacities. Transcript levels of different SODs located at various cellular compartments were affected depending on the place of action. Gene expression of CAT in leaves and roots was also affected after uranium exposure. Results indicate that oxidative stress plays an important role in uranium toxicity but suggest that plant responses differ for leaves and roots.

BACKGROUND: Few population studies have reported on the long-term changes in the internal cadmium dose and simultaneously occurring mortality. OBJECTIVE: We monitored blood cadmium (BCd), 24-hr urinary cadmium (UCd), and mortality in an environmentally exposed population. METHODS: Starting from 1985, we followed BCd (until 2003), UCd (until 1996), and mortality (until 2007) among 476 and 480 subjects, randomly recruited from low- exposure areas (LEA) and high-exposure areas (HEA). The last cadmium-producing plant in the HEA closed in 2002. RESULTS: From 1985-1989 to 1991-1996, BCd decreased by 40.3% and 18.9% in the LEA and HEA, respectively (p < 0.0001 for between-area difference). From 1991-1996 until 2001-2003, BCd remained unchanged in the HEA (+ 1.8%) and increased by 19.7% in the LEA (p < 0.0001). Over the entire follow-up period, the annual decrease in BCd averaged 2.7% in the LEA (n = 258) and 1.8% in the HEA (n = 203). From 1985-1989 to 1991-1996, UCd fell by 12.9% in the LEA and by 16.6% in the HEA (p = 0.22), with mean annual decreases of 2.7% (n = 366) and 3.4% (n = 364). Over 20.3 years (median), 206 deaths (21.5%) occurred. At baseline, BCd (14.6 vs. 10.2 nmol/L) and UCd (14.1 vs. 8.6 nmol/24-hr) were higher in deaths than in survivors. The risks (p < or = 0.04) associated with a doubling of baseline UCd were 20% and 44% for total and noncardiovascular mortality, and 25% and 33% for a doubling of BCd. CONCLUSIONS: Even if zinc-cadmium smelters close, historical environmental contamination remains a persistent source of exposure. Environmental exposure to cadmium increases total and noncardiovascular mortality in a continuous fashion without threshold. ispartof: Environmental health perspectives vol:116 issue:12 pages:1620-1628 ispartof: location:United States status: published

It is known that rhizospheric and endophytic bacteria may carry out a variety of functions improving plant growth (Mastretta et al., 2007). In agriculture they are under investigation as an alternative for chemical fertilizers to improve plant yield. These bacteria can also improve the nutrient status of their host plant due to biological nitrogen fixation (diazotrophy) and increasing nutrient availability into the rhizosphere through solubilization of minerals. Plant biomass production can also be increased via the synthesis of plant hormones by the bacteria. Plantassociated bacteria further seem to be able to reduce or prevent the deleterious effects of phytopathogenic organisms through competition, antibiosis and Induced Systemic Resistance (ISR) in plants. During recent years, their ubiquitous existence combined with the large variability of metabolic functions that they may carry out, attracted attention for their potential to improve phytoremediation. This thesis aims to improve our understanding of the potential contribution of plant-associated bacteria, which naturally occur in Nicotiana tabacum grown on soils with increased metal contents, to improve metal uptake and translocation in their host plant. We compared the endophytic consortia associated with tobacco plants growing on two metal contaminated sites....

Phytoremediation, the use of plants for the in situ treatment of contaminated soils and sediments, is an emerging technology that promises effective and inexpensive clean-up of certain contaminated sites. Phytoremediation is most suited for sites with shallow contamination (

Most biospheric nitroaromatic compounds (NACs) are industrial chemicals, such as explosives, dyes, polyurethane foams, herbicides, insecticides, and solvents, that often are recalcitrant to biological treatments and persist in the environment, constituting a hazard due to their toxicity and mutagenicity. In the present study, we evaluated perspectives for bioremediation and phytoremediation strategies at a nitroaromatic-contaminated site. Our research focussed mainly on 2,4-DNT and TNT. ...

This study examines the biological aspects related to alternative remediation strategies for Pb contaminated soils: EDTA induced Pb phytoextraction and amendment induced immobilization of soil Pb by means of inorganic soil amendments. The physiological effects of Pb-EDTA and EDTA were studied on bean plants (Phaseolus vulgaris L. Limburgse vroege), grown under strictly controlled conditions on a Hoagland nutrient solution. Addition of Pb-EDTA to the growth medium increased the capacity of enzymes belonging to three different compartments of defence mechanisms against oxidative stress in roots and primary leaves, before effects on plant morphology or chlorophyll content were observed. Pb-EDTA further drastically reduced Ca, Fe, Mn and Zn content in primary leaves. EXAFS analysis revealed that only 60-70% of all Pb in leaves was complexed to EDTA. Our data did not permit to conclude whether the observed phytotoxic response is due to the presence of Pb-EDTA, unchelated Pb or their combination. SPOD appeared to be an early biomarker for Pb-EDTA induced stress, possibly due to its role in cell wall lignification. Addition of EDTA alone decreased Fe, Mn, and Zn content in primary leaves of bean plants but did not induce any stress reaction. ...