plantago major research paper

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• Published: 23 December 2019

A protocol for systematic review of Plantago major L. effectiveness in accelerating wound-healing in animal models

• Fernanda de Cássia Israel Cardoso 1 ,
• Priscila Peruzzo Apolinário 1 ,
• Jéssica da Silva Cunha Breder 1 ,
• Thalita Paranhos 1 ,
• Henrique Ceretta Oliveira 1 ,
• Ariane Dini Polidoro 1 ,
• Ana Railka Souza Oliveira Kumakura 1 &
• Maria Helena Melo Lima   ORCID: orcid.org/0000-0001-6521-8324 1  

Systematic Reviews volume  8 , Article number:  337 ( 2019 ) Cite this article

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Studies have indicated that Plantago major L. ( P. major ) has therapeutic properties, such as anti-inflammatory, antioxidant, antifungal, immunostimulatory, and tissue regeneration. This plant species is assumed to provide potent tissue repair and healing in treatments of skin wound injuries, but the understanding of its effectiveness is still unclear. The systematic review proposed herein aims to assess effectiveness of P. major for wound healing in animal models.

We will conduct database searches in BVS, PubMed, Scopus, Web of Science, CINAHL, and CABDirect. Reviewers will independently evaluate titles, abstracts, and full-text articles retrieved from databases to identify potentially eligible studies. Relevant articles will be assessed for risk of bias and quality. The database searches will include analysis of wound healing rate through macroscopic evaluation, photo images, or calculation of the wound area retraction until the wound closure. Relevant data will be compiled for the capability and effectiveness of P. major treatments in accelerating wound healing. Random effects meta-analysis models will be employed to compare among groups based on outcome variables from studies reporting sufficient high-quality data.

Results of this systematic review will be presented in a narrative synthesis form. They will provide a summary and clear understanding of the relevant current questions and evidences directly related to P. major effective tissue repair and healing. Outcomes of this systematic review will contribute with important information that could benefit future research efforts and potential applicability in humans.

Systematic review registration

PROSPERO CRD42019121962

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Because of their therapeutic effects, medicinal plants are used worldwide to treat many diseases. They are rich sources of phytochemicals with potential therapeutic effect in treatments using direct application of raw material. Moreover, they also play a role for the development of new medicinal drugs. Plantago major L. is one of the most abundant and widely distributed medicinal plant in the world. It is a perennial plant species that belongs to the genus Plantago and the family Plantaginaceae [ 1 ]. P. major leaves and seeds are reported to have analgesic, anti-inflammatory, antioxidant, immunomodulatory, antifungal, anticancer, and wound healing [ 2 , 3 , 4 ]. Medicinal benefits of P. major may be related to various bioactive compounds, such as flavonoids, alkaloids, terpenoids, phenolic compounds, iridoid glycosides, fatty acids, polysaccharides, and vitamins [ 2 , 5 ]. Recent studies have shown successful treatment of cutaneous wounds with certain plant species or natural substances isolated from plants [ 6 , 7 , 8 ]. P. major has been described to be effective for tissue repair and skin wound healing, but the extent of its effectiveness has not been evaluated.

First reports related to therapeutic uses of this plant species date from the twelfth and thirteenth centuries [ 9 , 10 ]. Nowadays, several researches using in vivo, in vitro, and ex vivo techniques have demonstrated potential healing activity of ethanol- and water-based extracts from P. major leaves and seeds [ 11 , 12 , 13 , 14 ]. To date, a systematic review on therapeutic effectiveness of this plant species on skin wound healing was not identified in the literature. Therefore, the findings of this proposed systematic review will significantly contribute to the current knowledge of the mechanism of action of P. major in the process of cutaneous wound healing.

According to the literature, P. major has various medicinal applications without any serious adverse effects. Besides, this plant species is widely distributed in many countries (11-14). These evidences encourage further search on the effectiveness of P. major on healing processes, as well as transfer of knowledge from research to clinical practice. Thus, the objective of this systematic review is to identify, select, and evaluate high-quality published research on the effectiveness of P. major in the healing process of cutaneous wounds.

Study question

What is the evidence in the literature for the effectiveness of using P. major for wound healing in animal models?

Protocol and registration

This protocol was registered on PROSPERO (CRD42019121962). The bias of the assessed experimental studies will be evaluated by the Systematic Review Center for Laboratory Animal Experimentation (SYRCLES) [ 15 ]. The quality of the studies will be evaluated by the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) [ 16 ]. This systematic review will search for primary studies in animal models with cutaneous wounds topically treated with P. major in comparison with the placebo/vehicle control group.

Electronic search methods for study identification

Search will be conducted in 7 electronic bases listed below alongside with their respective strategies: BVS : (“Wound Healing”) OR (Regeneration) AND (“Plantago major” OR “Plantago officinarum”). PubMed : (Wound Healing) OR “Wound Healing”) OR Healing, Wound) OR “Healing, Wound” OR Healings, Wound OR “Healings, Wound”) OR Wound Healings) OR “Wound Healings”) OR (Regeneration) OR Regeneration) OR Regenerations) OR Regenerations) AND (“Plantago major”) OR “Plantago officinarum”). Scopus : (“Wound Healing” OR “Healing, Wound” OR “Healings, Wound” OR “Wound Healings”) OR (TITLE-ABS-KEY (regeneration OR regenerations) AND (TITLE-ABS-KEY (“Plantago major” OR “Plantago officinarum”). Web of Science : (“Wound Healing” OR “Healing, Wound” OR “Healings, Wound” OR “Wound Healings”) (Regeneration OR Regenerations) (“Plantago major” OR “Plantago officinarum”). Embase : (“plantago major” OR “plantago major” OR “plantago major”) AND (“wound healing”/exp OR “wound healing”/syn OR “wound healing” OR “regeneration”/ OR “regeneration” OR “regeneration”). CINAHL : (“Wound Healing”) OR “Wound Healing” OR “Healing, Wound” OR “Healings, Wound” OR “Wound Healings” OR (“Regeneration”) OR Regeneration OR Regenerations AND “Plantago major” OR “Plantago major” OR “Plantago officinarum”. CABDirect : (Regeneration OR Regenerations) OR (“Wound Healing” OR “Healing, Wound” OR “Healings, Wound” OR “Wound Healings”) AND (“Plantago major” OR “Plantago officinarum”. Grey literature will not be included in the search.

Procedure for study selection

At first, titles and abstracts will be examined by two reviewers (FCIC and PPA) and then selected according to the criteria for potentially eligible studies. Duplicated studies will be excluded from the search. Possible discrepancies between the two reviewer evaluations will be discussed and resolved or decided by a third member (APD). Subsequently, studies that were identified as eligible will be submitted to a text review performed by two reviewers (TP and JBC). Disagreements among the text reviewers will be resolved by a third member (ARSO-K). The final list of publications to be included in the systematic review will be decided in plenum. The following data will be extracted: title, author, year, journal, study type, wound kind, formulation type, treatment time, method, comparison groups, and outcome (Table 1 ). Remaining discrepancies will be resolved in agreement with a third author, and then, they will be revised in plenum. Data will be extracted to a Microsoft Office Excel document.

Statistical Analysis will be performed by HCO, ARSO-K, and MHM on data collected from January 2006 to January 2019.

Criteria for inclusion and exclusion of studies

Studies selected for the systematic review must (a) be written in English; (b) use the animal model specifically in rats, mice, and rabbits; (c) include both genders; (d) and focus on acute or chronic cutaneous wound models. Studies must describe (a) the initial and final wound size, (b) the number of animals per group, (c) the time of treatment, (d) the present the concentration, (e) the formulation used in the treatment of the wound, (f) data about the wound-healing rate, (g) and cell markers that can be modulated by the therapeutic treatment, assessed by Morphometric analysis. Details are shown in Table 1 . Studies using P. major mixed with another medicinal plant species will be excluded, as well as the ones based on in vitro experimentation.

Intervention

The intervention group must include topical treatment with P. major . Treatment must describe formulation, concentration used, and initial and final size of the wound.

Quality assessment

Studies will be evaluated by two independent reviewers with the quality evaluation instrument CAMARADES, a 10-item checklist in which one point is granted for each question [ 10 ]. The analysis of results will use the SYRCLE’s RoB [ 9 ] instrument, which consists of 10 “yes,” “no,” or “not clear” questions, indicating low, high, and not clear risk of bias, respectively.

Comparators

Control group submitted to topical treatment with placebo/vehicle must be compared against the group receiving the analyzed intervention.

The outcome includes analysis of wound healing rates through macroscopic evaluation, photo images, or calculation of wound retraction area until wound closure.

Data synthesis

The results of this systematic review will be presented in the narrative synthesis form. Data on animal species, wound type, treatment time, and results of interventions will be tabulated in order to support findings of the search. Random effects meta-analysis models will be employed to compare among groups, based on outcome variables. Studies included in the review must disclose sufficient high-quality data. Weighted mean differences among groups with their respective 95% confidence intervals and p values will be presented in forest plots. The presence of heterogeneity will be evaluated by I 2 and chi-square statistical analyses. Funnel graphics will be constructed to evaluate publication bias.

To our knowledge, this is the first review to date evaluating effectiveness of P. major , a medicinal plant species, in wound treatments. This systematic review will be done solely for the English idiom due to financial constraints since it did not receive financial support. This is a limitation however attenuated by the fact that nowadays the vast majority scientific research is published in English. Results indicating efficacious and accelerated healing process using P. major topic treatment have been described in the literature. Even though efficiencies of this plant species in cutaneous healing process is not yet clear. Healing rates will be evaluated in cutaneous wounds that received P. major topical treatment compared with wounds that received placebo/vehicle treatment. P. major extract concentration, best response, time of use, and cell markers that can be modulated by the treatment are important variables to assess treatment effectiveness. Data on these variables might provide steady information; thus, they will be searched, compiled, and analyzed.

The results of this systematic review may contribute to the transferring of knowledge from P. major scientific research into clinical practice guidelines. Moreover, this systematic review will contribute with important information that could benefit future research efforts and potential applicability in humans.

Availability of data and materials

Not applicable

Abbreviations

Virtual Health Library

Centre for Agriculture and Bioscience International Bioscience International

Collaborative Approach to Meta-Analysis of Animal Data from Experimental Stroke

Enzyme-linked immunosorbent assay

Excerpta Medica Database

Cumulative Index to Nursing and Allied Health Literature

International Prospective Register of Systematic Reviews

Public/Publisher MEDLINE

SciVerse Scopus

Systematic Review Center for Laboratory animal Research

Medical Literature Analysis and Retrieval System

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Fernanda de Cássia Israel Cardoso, Priscila Peruzzo Apolinário, Jéssica da Silva Cunha Breder, Thalita Paranhos, Henrique Ceretta Oliveira, Ariane Dini Polidoro, Ana Railka Souza Oliveira Kumakura & Maria Helena Melo Lima

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Contributions

This study was conceptualized by MHML, PPA, and FCIC for developing the search strategy. TP, FCIC, and PPA completed the search and outlined the protocol. APD, ARSO-K, JCB, and HCO critically appraised the protocol and contributed to its development by revising subsequent versions. MHML, ARSO-K, and HCO will contribute equally to data collection and analysis and interpretation of the review. All authors will read, critically revise, and approve the review in a final manuscript.

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Correspondence to Maria Helena Melo Lima .

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de Cássia Israel Cardoso, F., Peruzzo Apolinário, P., da Silva Cunha Breder, J. et al. A protocol for systematic review of Plantago major L. effectiveness in accelerating wound-healing in animal models. Syst Rev 8 , 337 (2019). https://doi.org/10.1186/s13643-019-1255-6

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Immunoenhancing properties of Plantago major leaf extract

Affiliation.

• 1 Department of Biomedical and Therapeutic Sciences, Section of Medical Sciences, University of Illinois College of Medicine, Peoria, IL 61656-1649, USA. [email protected]
• PMID: 11113999
• DOI: 10.1002/1099-1573(200012)14:8<617::aid-ptr674>3.0.co;2-n

Plantago major (PM), also known as plantain, is a weed found in temperate zones worldwide. PM leaves have been associated with various biological properties ranging from antiinflammatory, antimicrobial and antitumour to wound healing. However, its mechanism of action associated with boosting of the immune function remains to be elucidated. We found that endotoxin-free methanol extracts from PM leaves, at doses of 50, 100, 250, and 500 microg/mL, were associated with 4.4 +/- 1, 6 +/- 1, 12 +/- 0.4, and 18 +/- 0.4-fold increases of nitric oxide (NO) production, and increased TNF-alpha production (621 +/- 31, 721 +/- 36, 727 +/- 36, and 1056 +/- 52 U/mL, respectively) by rat peritoneal macrophages, in the absence of IFN-gamma or LPS. NO and TNF-alpha production by untreated macrophages was negligible. In addition, PM extracts potentiated Con A-induced lymphoproliferation (3- to 12-fold increases) in a dose-dependent fashion, compared with the effect of Con A alone. The regulation of immune parameters induced by plant extracts may be clinically relevant in numerous diseases including chronic viral infections, tuberculosis, AIDS and cancer.

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Article Contents

1. introduction, 2. materials and methods, 4. discussion, supplementary data, 5. contributions, conflict of interests, data availability statement.

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Genome assembly of the pioneer species Plantago major L. (Plantaginaceae) provides insight into its global distribution and adaptation to metal-contaminated soil

These authors contributed equally to this work.

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Shanwu Lyu, Qiming Mei, Hui Liu, Baosheng Wang, Jun Wang, Hans Lambers, Zhengfeng Wang, Bin Dong, Zhanfeng Liu, Shulin Deng, Genome assembly of the pioneer species Plantago major L. (Plantaginaceae) provides insight into its global distribution and adaptation to metal-contaminated soil, DNA Research , Volume 30, Issue 4, August 2023, dsad013, https://doi.org/10.1093/dnares/dsad013

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Plantago is a major genus belonging to the Plantaginaceae family and is used in herbal medicine, functional food, and pastures. Several Plantago species are also characterized by their global distribution, but the mechanism underpinning this is not known. Here, we present a high-quality, chromosome-level genome assembly of Plantago major L., a species of Plantago , by incorporating Oxford Nanopore sequencing and Hi-C technologies. The genome assembly size was approximately 671.27 Mb with a contig N50 length of 31.30 Mb. 31,654 protein-coding genes were identified from the genome. Evolutionary analysis showed that P. major diverged from other Lamiales species at ~62.18 Mya and experienced two rounds of WGD events. Notably, many gene families related to plant acclimation and adaptation expanded. We also found that many polyphenol biosynthesis genes showed high expression patterns in roots. Some amino acid biosynthesis genes, such as those involved in histidine synthesis, were highly induced under metal (Ni) stress that led to the accumulation of corresponding metabolites. These results suggest persuasive arguments for the global distribution of P. major through multiscale analysis. Decoding the P. major genome provides a valuable genomic resource for research on dissecting biological function, molecular evolution, taxonomy, and breeding.

Plantago is a large genus within the Plantaginaceae family, including over 250 species, with broad geographic distributions in temperate and high-elevation tropical regions. 1 , 2 Although the Plantago genus is well characterized from a taxonomic perspective, its intrageneric classification is still controversial and inadequate, especially within the subgenus Plantago , largely due to the plesiomorphic characters, low morphological variation, and lack of a reference genome. 1 , 3 Some Plantago species have cosmopolitan distributions and several are used as herbal medicine, food ingredients, and in pastures, such as Plantago major . 4 , 5

Plantago major , broadleaf plantain or greater plantain, is diploid (2 n = 12), wind-pollinated, and self-compatible. 1 , 6 It is a perennial herbaceous plant with a fibrous root system, a rosette of oval-shaped leaves, and several long spike inflorescences. It can be found in soils with a wide range of fertility, pH, temperature, and moisture and shows outstanding tolerance to diseases, pests, radiation, and chemical pollution. 7 , 8 P. major is native to central, northern and southwest Asia, and Europe and naturalized worldwide. 9 It grows in various habitats, including meadows, wastelands, roadsides, and other sites of anthropogenic disturbance. 9

To develop resistance and adaptation, plants have evolved with a series of responding mechanisms such as DNA repair, plant–pathogen interaction, and metabolic changes. Metabolic changes include synthesis and accumulation of metabolites. Such specialized metabolism may confer plants with stress resistance and involve the production of primary metabolites such as amino acids and nucleic acids and secondary metabolites such as phenolics and flavonoids. 10 These specialized metabolites may not directly play a part in plant growth and development, but they are essential in interacting with the environment for adaptation and defense. In the case of primary metabolites, some amino acids (e.g. proline, histidine, and glutamic acid) can act either as signaling molecules or as chelators dealing with stresses such as drought, salt, and metal stresses or as precursors in the biosynthesis of secondary metabolites. 11 , 12 Even more so with secondary metabolites, in general, ecological and environmental disturbance can induce their accumulation. 13 , 14 Polyphenols are an excellent example of secondary metabolites elevating plant adaptation: because these molecules are involved in drought, high soil salinity, extreme temperature, UV-irradiation, nutrient deficiencies, metals, and pathogen attacks. 15

Plantago contains numerous secondary metabolites, such as phenolic compounds and flavonoids. 11 , 16 Some free amino acids and polyphenols can activate plant tolerance to drought and extreme temperature, defense against pathogen attack, inhibition of DNA damage, and chelation of metal ions. 15 , 17 , 18 P. major has the potential as a pioneer in phytoremediation of contaminated soil by accumulating metal ions (e.g. Cu, Pb, Zn, Cd, Cr, and Ni) 19 , 20 that may be based on its production of certain specialized metabolites. P. major is also a model plant for vasculature biology study since vascular tissue can be isolated easily and intactly from mature leaves. 21 A handy and efficient transformation method for P. major was also developed, 22 which can implement functional verification in situ . Based on these applications, the transport of both nutrient and information molecules was well characterized, such as the transporters of sucrose, 23 the responses to salinity, 24 and the responses under phosphate (Pi) deficiency. 25

To date, the chemical compounds, ecology, and population genetics of P. major have been widely studied. 26–28 However, the molecular mechanisms underlying its high pollution tolerance and broad fitness in diverse environments are largely unknown. A high-quality genome assembly is necessary to address these questions. We conducted a chromosome-level genome assembly of P. major and found that genes accounting for the biosynthesis of specialized metabolites, such as free amino acids and polyphenols, were expanded. The genes related to polyphenol biosynthesis are more highly expressed in roots. Another expanded gene family, the histidine biosynthetic (HISN) gene family, which correlates strongly with Ni tolerance, has been characterized. The expression patterns of PmHISN genes provided clues for the tolerance of this species to Ni. These genomic data will provide clues for elucidating the molecular mechanism underlying the robust adaptation of P. major to diverse environments. The reference genome will be a valuable resource for genetic studies and improvement of Plantago, such as genome-assisted breeding of novel cultivars with low-level heavy metal ions.

2.1 . Sequencing, genome size estimation, and assembly

One individual of P. major (PlanMa1, Fig. 1a ) was provided by the Shennong Caotang Museum of Traditional Chinese Medicine in Guangzhou, China (113.3445 E, 23.2029 N). It is an inbred line that descended from a single seed for six generations. P. major and its seeds as Chinese herbs have the effect of clearing heat, diuretic and laxative. The total genomic DNA was isolated from fresh leaves using a DNA extraction kit (QIAGEN, Hilden, Germany). Three Nanopore libraries with insert sizes larger than 20 kb were constructed according to a standard protocol (Oxford Nanopore Technology, Oxford, UK), followed by single-molecule DNA sequencing. The libraries were sequenced with flow cells on the PromethION platform (Oxford Nanopore Technology, Oxford, UK). A total of 130.60 Gb (~180× of the estimated genome size) read bases were generated. Adapters and low-quality reads ( Q ≤ 15) were removed from datasets. Before genome assembly, we estimated the genome size utilizing the K -mer method. The number of 17-mer sequences was counted by KmerFreq as included in SOAPdenovo package v2.04. 29 The P. major genome size was estimated by the following formula: G = K num / K depth , where the K num refers to the total number of K -mers, and K depth is the most frequent peak. Sequencing data were assembled using NextDenovo v1.0 (read_cuoff = 1k and seed_cutoff = 20k, blocksize = 8g) and corrected by NextPolish v1.0.1 with default parameters. 30 The genome was assembled using the following parameters: nextgraph_options= -n 83 -Q 6 -I 0.64 -S 0.27 -N 2 -r 0.48 -m 3.81 -C 1180183 -z 20. The quality of genome assembly was assessed by BUSCO v5.3.2. 31 The genome assembly was assessed using next-generation sequencing. The sequence map rate is 99.02% and the coverage rate is 90.83% and the final accuracy of the genome is 99.99%.

De novo genome assembly and annotation of P. major . (a) Morphological characteristics of P. major include seeds, seed pods, seedlings, and adult plants. Black scale: 2 mm; white scale: 4 cm. (b) Contact map of Hi-C-based intra-chromosomal interactions. (c) Genetic collinearity among six P. major chromosomes. (a) chromosome length; (b) gene density; (c) gene expression pattern in leaf; (d) gene expression pattern in root; (e) gene expression pattern in seed; (f) GC content; and (g) paralogous gene pairs.

2.2 Hi-C assembly

Fresh leaf material was fixed in formaldehyde to give DNA–protein bonds. The restriction enzyme Dpn II (New England Biolabs, Hitchin, UK) was used to digest the chromatin. The 5ʹ overhang ends were filled in with biotinylated residues. After re-ligation, DNA was sheared into ~350 bp fragments by sonication. The Hi-C library was prepared following a standard procedure and sequenced on the Illumina NovaSeq 6000 platform with PE150 mode (Illumina, San Diego, USA). A total of 903.68 million clean Hi-C paired-end reads were mapped to genome assembly using Bowtie2 v2.3.2 (-end-to-end model, parameters: --very-sensitive, -L 30). 32 LACHESIS (CLUSTER MIN RE SITES = 100, CLUSTER MAX LINK DENSITY = 2.5, CLUSTER NONINFORMATIVE RATIO = 1.4, ORDER MIN N RES IN TRUNK = 60, ORDER MIN N RES IN SHREDS = 60) assembly was conducted to cluster Hi-C contigs into chromosome groups. 33 The number of pseudo-chromosomes was set to six according to previous karyotyping studies. 34 Then the genome was divided into 100 kb bins. A matrix was constructed based on the pairwise comparison by Hi-C-Pro v2.11.1, 35 and a contact map was plotted to estimate the quality of pseudo-chromosome using the ggplot2 v3.3.6 package as implemented in R. 36

2.3 De novo genome annotation

Repetitive elements were annotated and masked in the P. major final genomic assembly using de novo and homology-based methods. First, simple sequence repeat (SSR) sequences were identified by MISA-web. 37 Also, LTR_FINDER, 38 MITE-Hunter 39 as well as RepeatModeler v1.0.11 ( http://www.repeatmasker.org/RepeatModeler/ ) were used to construct repeat libraries. Then, libraries were combined with Repbase database ( https://www.girinst.org/repbase/ ). Finally, the repeat regions were predicted using RepeatMasker v4.0.7 ( http://repeatmasker.org/cgi-bin/WEBRepeatMasker ). Also, we annotated non-coding RNAs (ncRNA) in the P. major genome by blasting the Pfam database ( http://pfam-legacy.xfam.org/ ), tRNAscan-SE ( http://lowelab.ucsc.edu/tRNAscan-SE/ ), and RNAmmer ( https://services.healthtech.dtu.dk/service.php?RNAmmer-1.2 ).

Gene structural annotation of P. major was performed following three strategies: (i) de novo prediction performed by AUGUSTUS v3.3.3 40 and Fgenesh v2.1 41 ; (ii) homology-based annotation using GeMoMa v1.6.4 42 ; and (iii) finding coding regions in transcripts by PASA v2.5.2 43 and TransDecoder v5.5.0 ( https://transdecoder.github.io/ ). Results were combined by EVidenceModeler v1.1.1 ( http://evidencemodeler.github.io/ ). Then, genes containing transposable elements were removed by TransposonPSI ( http://transposonpsi.sourceforge.net ).

The predicted genes were further functionally annotated. Sequences queried against databases including Gene Ontology (GO) ( http://geneontology.org/ ), KEGG ( https://www.genome.jp/kegg/ ), KOG (ftp://ftp.ncbi.nih.gov/pub/COG/KOG/), non-redundant (NR) (ftp://ftp.ncbi.nih.gov/blast/db) and Swissprot ( https://www.uniprot.org/help/downloads/ ) by blastp ( e -value < 1e–10). In addition, P. major protein sequences were compared with protein domain annotations of all available databases using InterProScan v5.32-71.0. 44 BUSCO v5.3.2 was used to validate the correction of gene annotations with the embryophyta_odb10 and default parameters. 31

2.4 Evolutionary analysis

The genomes of 14 eudicots, including Olea europaea, Sesamum indicum, Dorcoceras hygrometricum, Striga asiatica, Salvia splendens, Handroanthus impetiginous, Erythranthe guttata, Genlisea aurea, Mikania micrantha, Fragaria vesca, Abrus precatorius, Ipomoea nil, Ricinus communis , and Arabidopsis thaliana were used in the phylogenetic analysis. A monocot genome ( Setaria viridis ) was used as an outgroup for the phylogenetic analysis. OrthoFinder v2.5.4 [57] was utilized to detect orthologous groups of the P. major genome using an e -value threshold of 1 e– 10. 209 orthogroups with a minimum of 75% of species having single-copy genes were used for phylogeny reconstruction. The protein sequences were aligned using MAFFT v7.508, 45 after which the resulting multiple sequence alignment (MSA) datasets were converted to coding DNA sequence (CDS) format using PAL2NAL version 14.1. 46 Sites of poor alignment quality were removed by Gblocks v 0.91b. 47 The final dataset was generated by concatenating alignments. The phylogenetic tree was constructed by RAXML v8.2.11 with the GTR model and gamma distribution. 48 The RelTime-ML, implemented in MEGA X software, was used for an evolutionary time estimate. 49 Fossil records from the Timetree of Life 50 were used to calibrate the inferred tree. The CAFÉ v5.0.0 package was used to investigate the expansion and contraction of gene families. 51 Enrichment analysis of KEGG was performed on unique genes and expansion gene families.

The 4DTv (four-fold synonymous third-codon transversion) method was used to assess the WGD (Whole-genome duplication) event of P. major as well as two Lamiales ( O. europaea and S. indicum ). An all-to-all search was performed by blastp ( e -value < 1e–10). Collinear regions were identified by MCscan among these three genomes. 52 Also, the synonymous substitution rates ( Ks ) of collinear regions were calculated using CodeML in the PAML package v4.8. 53 Paralogous gene pairs, together with the gene density, gene expression pattern (leaf, root, and seed), and GC content were visualized using the R package ‘circlize’. 54

2.5 Distribution and ecological ranges

For analyzing the distribution and ecological ranges of P. major and three other globally distributed species ( I . nil, M . micrantha , and S . viridis ), we first obtained geo-referenced locality data for each species from the GBIF (Global Biodiversity Information Facility) (GBIF, https://www.gbif.org ) in R, and the occ_download function in the rgbif package ( https://CRAN.R-project.org/package=rgbif ). We checked each species’ occurrence to ensure the database was representative of their distributions. For each locality, we extracted the aridity index (AI = mean annual precipitation/potential evapotranspiration; MAP/PET) using the Global-Aridity dataset ( https://cgiarcsi.community/data/global-aridity-and-pet-database/ ). Next, we extracted soil nitrogen concentration and soil bulk density for the 0–20 cm soil depth from the World Soil Database 55 at 1 × 1 degree resolution. To compare different ecological ranges among species, we used ANOVA and multiple comparisons (Tukey HSD) based on the mean and variance of each environmental factor across the whole range.

2.6 Transcriptome sequencing

The PlanMa1 plants were cultivated in pots filled with a substrate mixture of peat moss and perlite in a 3:1 ratio. The plants were maintained under constant conditions of 26°C temperature and a photoperiod of 16 h of light followed by 8 h of darkness. Total RNA of 40-day-old seedlings was extracted from three replicates of fresh leaves, roots, and seeds and treated with DNase I (QIAGEN Genomic). The RNA integrity was validated using the NanoDrop One UV–Vis spectrophotometer. Then mRNAs were enriched by Oligo (dT)-attached magnetic beads and random hexamers were used for cDNA synthesis. RNA-sequencing libraries were subsequently sequenced on the Illumina HiSeq platform with PE150 mode. Raw data were filtered by fastp v0.12.6. 56 The FPKM (Fragments per kilobase per million mapped reads) was used to estimate the expression level of transcripts. In this study, we focus on the expression of polyphenol synthesis genes, and FPKM values were calculated by StringTie v2.1.7. 57 Also, differential gene expression analysis was conducted using DESeq2 with fold change ≥ 2 and FDR-adjusted P -value ≤ 0.05. 58

2.7 PmHISNs identification and expression pattern analysis

Arabidopsis HISN protein sequences were used as queries to perform BLASTP searches to the P . major database with the e -value<1e–10. Only those with e -value<1e–100 were kept as candidates. A phylogenetic tree was drawn by MEGA X 49 with the Maximum Likelihood method and the bootstrap value of 1,000 using the protein sequences of AtHISNs and PmHISNs.

The P. major (PlanMa1) seeds were sown in soil in pots and subsequently maintained in a controlled greenhouse with 16 h light and 8 h darkness periods at 25°C. After 35 days, the experimental P. major seedlings were transplanted into a half-strength Hoagland solution, which was replaced on alternate days. Following an additional week, the seedlings were transferred to fresh half-strength Hoagland solutions that were supplemented with varying concentrations of NiSO 4 (0 μM, 200 μM, and 500 μM) for a duration of 24 h. Plants were harvested and split into roots and shoots, with the root material being washed with double-distilled water to remove any residual Ni ions.

All samples intended for qRT-PCR were snap-frozen in liquid nitrogen and subsequently stored at –80°C until required. RNAs were extracted as previously mentioned and reverse-transcribed using an oligo (dT) primer in combination with SuperScript II reverse transcriptase (Vazyme). qRT-PCR was performed in the Quantagene™ q225 Detection System, using SYBR Green Master Mix reagent (Vazyme) according to the manufacturer’s instructions. PmACTIN2 served as the internal control, and gene expression levels were determined using 2 ˉΔΔCt method. 59 A list of the primers used is provided in Supplementary Table S1 . The organ-specific expression patterns of AtHISN1A and AtHISN1B were obtained from the TAIR database ( https://www.arabidopsis.org/ ).

2.8 Free amino acids measurement

For the analysis of free amino acids (FAA), all samples, including leaves and roots, were subjected to drying in an oven set to 80°C for a duration of 24 h. 0.50 g of each sample was extracted utilizing 25 ml of 0.01 M HCl for 30 min, at ambient temperature. Following centrifugation, 2 ml of supernatant was transferred into new tubes and combined with equal volumes of an 8% (v/v) sulfosalicylic acid solution. The resultant mixtures were centrifuged at 12,000 rcf for 5 min. Finally, the supernatant was analyzed by Amino Acid Analyzer (Sykam S433, Eresing, Germany).

3.1. De novo assembly of P. major genome

A total of 96.66 Gb (Gigabases) Nanopore sequencing data were generated and used for further analysis ( Supplementary Table S2 ). The genome of P. major consists of six pairs of chromosomes (2 x = 12, n = 6), and its size is approximately 690 Mb (Megabases). 6 In this study, the genome size was estimated to be ~701 Mb based on K -mer analysis ( Supplementary Figure S1a ), and the final assembly was 671.27 Mb with a contig N50 size of 31.30 Mb ( Table 1 ). The longest contig reached 72.24 Mb. The quality of the genome assembly was assessed by BUSCO (Benchmarking Universal Single-Copy Orthologs). 31 We successfully detected 95.49% of the complete BUSCO (S + D) ( Supplementary Figure S1b and Table 1 ).

Statistics for the assembly of P. major genome

Based on Hi-C assembly with the agglomerative hierarchical clustering algorithm, 157 contigs containing 592.23 Mb Hi-C data were arranged and placed on six pseudochromosomes, representing 88.23% of total bases ( Table 1 ). The size of chromosomes ranged from 74.69 to 113.78 Mb. A contact map was plotted to validate the correction of the Hi-C assembly; the assembled six pseudochromosomes (named LG01–LG06) corresponded to the chromosome numbers in P. major ( n = 6) ( Fig. 1b ).

3.2. Annotation of P. major genome

Repetitive elements were annotated and masked in P. major before gene prediction. A total of 3.90 million SSR were detected ( Supplementary Table S3 ). The repeat elements of the P. major genome were estimated to be 469.83 Mb, corresponding to 69.99% of the genomic assembly ( Supplementary Fig. S1c and Supplementary Table S4 ). Non-coding RNAs (ncRNAs) were predicted in the genome as well ( Supplementary Table S5 ).

Protein-coding genes were annotated by integrating de novo homology-based and RNA-Seq-based results. The generated consensus P. major gene set included 31,654 protein-coding genes, and the average length of coding DNA sequences (CDS) was 1,184.4 bp. The mapping rate of RNA-Seq reads was 99.02% and the coverage rate of annotated genes was 90.83%. Functions of 28,390 genes were annotated, corresponding to 89.69% of the predicted genes ( Table 2 ). The quality of the annotation was assessed by BUSCO (Benchmarking Universal Single-Copy Orthologs). 31 We successfully detected 1,350 BUSCOs in the embryophyta_odb10 database, corresponding with 98.18% of P. major genes ( Supplementary Fig. S1b and Table 2 ). After gene annotation, we analyzed gene density, GC content, organ-specific gene expression patterns, and paralogous genes ( Fig. 1c ). There were 2048 (12.93%) pairs of tandem genes and 4685 (14.80%) collinear genes ( Supplementary Tables S6 and S7 ).

Statistics for functional annotation of P. major genome

3.3. Evolutionary analysis

The genome of P. major was compared with that of other angiosperms, including Striga asiatica, Salvia splendens , Erythranthe guttata , Handroanthus impetiginous , Sesamum indicum , Genlisea aurea , Dorcoceras hygrometricum , and Olea europaea, which are all Lamiales and thus phylogenetically closely related to P. major , and that of Ipomoea nil, Mikania micrantha, Ricinus communis, Arabidopsis thaliana, Abrus precatorius , and Fragaria vesca , which are globally widely distributed species. A monocot genome ( Setaria viridis ) was used as an outgroup in the phylogenetic analysis ( Fig. 2a and Supplementary Table S8 ). Among the total 31,654 annotated genes in the P. major genome, 4,480 genes from 810 families in the P. major genome were species-specific compared with the other 15 species ( Supplementary Table S8 ).

Evolutionary analysis of the P. major genome. (a) Phylogenetic tree of 16 angiosperms. Node and taxa labels are numbers of gene families manifesting expansion (green) and contraction (red) among 16 angiosperms. The evolutionary time scale is displayed below the tree. Distribution of 4DTV (b) and Ks (c) in three Lamiales. Two WGD events were observed in P. major (indicated as A and B). (d) Overlap of gene families in three Lamiales. (e) KEGG enrichment analysis of unique gene families in the P. major genome. (f) KEGG enrichment analysis of expansion gene families in the P. major genome.

Whole-genome duplication (WGD) is a prevalent phenomenon in angiosperm plants, providing a vast source of raw genetic material for gene genesis. In this study, we investigated genome expansion in P. major by analyzing WGD events. We estimated 4DTv and Ks values based on paralogous gene pairs within collinear regions identified in P. major , O. europaea , and S. indicum . Genome-wide doubling events generate numerous homologous genes, as reflected in the Ks values, which exhibit a large number of homologous gene pairs with closely clustered Ks values, and Ks peaks correspond to the occurrence of genome-wide doubling events. A larger number of gene pairs with 4DTV presence suggests greater genomic diversity or an increased number of redundant genes, possibly indicating species differentiation or ongoing genome duplication. Intra-genome collinearity analysis revealed sharp peaks in both 4DTV and Ks, confirming the occurrence of WGD events in P. major , O. europaea , and S. indicum ( Fig. 2b and c ). Furthermore, P. major 4DTV results exhibited two peaks, signifying two WGD events: one occurred during the early evolutionary stage of Lamiales (Peak A, 4DTV ≈ 0.55 and Ks ≈ 1.55), and the other was a more recent genome duplication, potentially occurring within Plantaginaceae (Peak B, 4DTV ≈ 0.35 and Ks ≈ 0.07) ( Fig. 2b and c ). In contrast, O. europaea and S. indicum had only one pronounced WGD event.

3.4. Unique and expanded genes enriched in metabolite biosynthesis and defense in P. major

A comparison of gene families was made among P. major, O. europaea , and S. indicum ( Fig. 2d ). The three species shared 8,881 out of the 15,930 orthologous gene families. There were 1,048 unique gene families in P. major , which shared more gene families with S. indicum (3,866) than with O. europaea (2,135) ( Fig. 2d ). KEGG enrichment analysis indicated unique families enriched in primary metabolite pathways such as histidine metabolism (map00340) and secondary metabolite pathways such as the phenylpropanoid biosynthesis pathway (map00940), isoflavonoid and flavonoid biosynthesis pathways (map00943 and map00941), and glutathione metabolism (map00480). Free histidine (His) can chelate Ni in plants and is responsible for nickel transport and tolerance. 60 Phenylpropanoids, isoflavonoids, and flavonoids can improve plants’ tolerance to environmental stresses. 14 Glutathione is essential for PCs (phytochelatins) synthesis, a principal class of metal chelators (Yadav 2010). Unique genes in DNA repair (nucleotide excision repair and mismatch repair, i.e. ko03420 and ko03430) and plant defense activities (plant–pathogen interaction, i.e. ko04626) were also enriched in P. major ( Fig. 2e ).

Compared with other angiosperms, 1,486/4,449 gene families manifested expanded/contracted patterns in P. major . The KEGG enrichment analysis of expansion showed similar results for unique families. Some expanded gene families were related to the biosynthesis of amino acids (map01230), histidine metabolism (map00340), the phenylpropanoid biosynthesis pathway (map00940), isoflavonoid and flavonoid biosynthesis pathways (map00943 and map00941), and glutathione metabolism (map00480) ( Fig. 2f ). These results indicate that those resistance genes retained after WGD events may allow P. major to exhibit a global distribution and adaptations, and the specialized metabolites may explain P. major ’s repertoire of habitat types and environmental conditions.

3.5. Adaptation strategy of P. major

Plantago major has widely naturalized throughout much of the world ( Fig. 3a ). Compared with three globally distributed species ( I . nil , M . micrantha , and S . viridis ), P. major occupies much larger ranges of climatic conditions and soil environments (the widest interquartile ranges in Fig. 3b–d ). Specifically, P. major grows over a wide climatic range, but moderately in arid environments (intermediate aridity index values, Fig. 3b ) and acidic or infertile soils (the most significant values of soil nitrogen concentration and the lowest values of soil bulk density; Fig. 3c and d ). The resistance genes mentioned above may contribute to P. major ’s adaptation to harsh conditions. Given its wide adaptation, P . major can be used as a pioneer plant in new assarts or disturbed lands.

Global distribution of P. major and ecological range comparisons with three other globally distributed herbaceous species. (a) Distribution localities of P. major . Red dots indicated regions of P. major distribution and the deeper of the color, the more of the P. major . (b–d) Comparisons of environmental factors among four globally distributed species: (b) aridity index (indicating water stress), (c) soil nitrogen concentration (indicating soil fertility), and (d) soil bulk density (indicating resistance to trampling). Sample sizes for each species are under each violin in (b). Data are based on climatic variables and soil properties across global distribution localities of each species. The violin shows data distribution and niche breadth of each species, while the black boxplot inside each violin indicates the median (white dot), first and third quartiles (upper and lower limits of the box), and the interquartile range (whiskers). Difference letters on top of each violin indicate significant differences based on multiple comparisons (Tukey HSD).

3.6. High expression of polyphenol synthesis genes contributes to P. major ’s global distribution

Polyphenols have properties of defense against biotic and abiotic stresses such as pathogen attacks, oxidants, and ultraviolet radiation. 61 We identified genes in P. major involved in polyphenol synthesis ( Supplementary Fig. S2 ). Most gene families involved in polyphenol metabolism were expanded, for example, cinnamoyl-CoA reductase (EC: 1.2.1.44) and 4-coumarate-CoA ligase (EC: 6.2.1.12).

To assess differences in gene expression patterns of phenylpropanoid biosynthesis genes among organs, we analyzed 78 differentially expressed genes (DEGs). The correlation of the expression patterns in different organs was relatively low, while replicates of the same organ showed a similar expression pattern ( Fig. 4a ). Moreover, the heatmap of the DEGs indicated that leaves, seeds, and roots elegantly exhibited quite different transcriptome profiles ( Fig. 4b ). Most genes showed a higher expression level in roots than in leaves and seeds, except for the K05350 bglB. Organ-specific analysis of DEG indicated that the expression level of phenylpropanoid biosynthesis genes was significantly higher in roots than in leaves and seeds ( Fig. 4c–e ). The numbers of up- and down-regulated genes in roots and leaves were 49 and 19, respectively. For root/seed and leaf/seed comparisons, the numbers of up- and down-regulated genes were 62/21 and 22/17. The results of our analysis suggest that the expression level of phenylpropanoid biosynthesis genes is significantly higher in roots compared to leaves and seeds in P. major . Our findings indicate that the plant would activate polyphenol synthesis in its roots, which may contribute to its global distribution.

Expression of phenylpropanoid biosynthesis genes. (a) Transcriptome correlations (Pearson’s correlation coefficients) across samples for each tissue for all annotated genes. (b) The heatmap of genes related to phenylpropanoid biosynthesis. Blue cells denote down-regulated DEGs, and red cells denote up-regulated DEGs. (c) Protein classes of DEGs and KEGG pathways enriched by up- (red) and down- (blue) regulated genes between leaves and roots. (d) Protein classes of DEGs and KEGG pathways between roots and seeds. (e) Protein classes of DEGs and KEGG pathways between leaves and seeds.

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Using Plantago major and Plantago lanceolata in environmental pollution research in an urban area of Southern Poland

Iryna skrynetska.

1 Department of Ecology, Faculty of Biology and Environmental Protection, University of Silesia, Bankowa 9, 40-007 Katowice, Poland

Jagna Karcz

2 Scanning Electron Microscopy Laboratory, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland

Gabriela Barczyk

Marta kandziora-ciupa, ryszard ciepał, aleksandra nadgórska-socha, associated data.

The aim of this study was to perform a complex assessment of changes in the elements of an ecosystem that are caused by environmental pollution in industrial and urban biotopes. The study focused on three sites: a park, a road and the site of the metallurgical plant “Pokój” in the city of Ruda Śląska (Southern Poland), which are each under a different level of anthropogenic load. Soil and plant material samples ( Plantago major and Plantago lanceolata leaves) were investigated by performing biochemical, ecophysiological and scanning electron microscopy (SEM) analyses. A significant difference was observed in all of the study samples. The content of Pb, Zn and Cd in the soil samples that had been collected at the site of the metallurgical plant exceeded the permitted limits (Cd > 4 mg kg −1 , Pb > 100 mg kg −1 , Zn > 300 mg kg −1 ). The content of Fe, Mn, Pb, Cd and Zn in the plant material was much higher in unwashed samples than in washed samples. The concentrations of potentially toxic elements (PTEs) were below the permitted level in the leaves of Plantago lanceolata for Cd (> 5 mg kg −1 ) and in the leaves of Plantago major for Zn (> 100 mg kg −1 ). The SEM observations revealed a significant decrease in the stomata pore length (SPL) in the Plantago lanceolata leaves that had been collected at the road site compared with the plants from the park site. The elemental content on the leaf surface was also determined using X-ray microanalysis. The total chlorophyll (Chl) content, ascorbic acid (AA), proline, guaiacol peroxidase (GPX) activity, pH, relative water content (RWC) and air pollution tolerance index (APTI) were evaluated. The APTI for the investigated species ranged from 5.6 to 7.4, which demonstrated that the studied plant species are sensitive to air pollutants.

Electronic supplementary material

The online version of this article (10.1007/s11356-019-05535-x) contains supplementary material, which is available to authorized users.

Introduction

Over the last several decades, the quality of the environment has undergone a significant deterioration, which was primarily due to rapid developments in industry as well as urbanisation. Environmental pollution has become a factor that is responsible for many negative effects on the health of fauna and flora as well as on the ecosystem as a whole because of potentially toxic metals that do not degrade and accumulate in the environment, most of which have long-term toxic effects on living organisms (Kardel et al. 2010 ; Remon et al. 2013 ; Muszyńska et al. 2018 ). However, the effects of this interaction on the function and structure of the elements of an urban ecosystem have not yet been adequately quantified and are poorly understood.

In flora, the epidermis is the first site of interaction with atmospheric pollution because pollutants first pass through the stomata of the epidermal tissues. The stomata, which regulate the flow of gases entering into or escaping out of leaves, are an excellent site to study the interaction between plants and their environment because they are the first to be affected by air pollution, which may cause changes in their morphology (Robinson et al. 1998 ; Kardel et al. 2010 ; Uka et al. 2017 ). There are many different biochemical and physiological mechanisms that help plants adapt to pollutants, and their efficiency can be assessed by a number of parameters such as the total chlorophyll (Chl) content, ascorbic acid (AA) content, pH and relative water content (RWC). All of these indexes make up the so-called air pollution tolerance index (APTI). The value of the APTI defines a plant’s tolerance to pollution because these parameters determine a plant’s adaptation to the environment and thus predetermine the sensitivity or resistance of a species to pollution (Lakshmi et al. 2008 ; Prajapati and Tripathi 2008 ). Additionally, a biochemical assessment of variations in metabolites could be helpful in defining the tolerance of a species. Proline accumulation is regarded as an indicator of heavy metal stress and enzymatic antioxidant components such as GPX may be used as an indicator of environmental stress for an ecosystem (Kandziora-Ciupa et al. 2017 ; Nadgórska-Socha et al. 2017 ).

The aim of this study was to perform a complex assessment of changes in the elements of an ecosystem that are caused by environmental pollution in industrial and urban biotopes. Two ruderal species, Plantago major and Plantago lanceolata , were selected for this study. The Plantago species has been used as a traditional medicinal plant in many parts of the world for centuries (Abd El-Gawad et al. 2015 ; Gomes de Andrade et al. 2018 ). Plantago lanceolata and Plantago major are easy to recognize and are very common in urban environments and in the countryside. Previous studies have indicated that the Plantago major and Plantago lanceolata species contain significant levels of trace elements (Tinkov et al. 2016 ; Nadgórska-Socha et al. 2017 ; Skrynetska et al. 2018 ).

The objective of this study was to perform a comparative analysis of selected ecophysiological and biochemical parameters and to determine the metal concentrations in soils and plants in samples that had been collected from three areas with different levels of the anthropogenic load. The data obtained enabled us to observe any differences in the morphology and physiological parameters, to analyse the air pollution tolerance indexes and to assess the potential use of the tested species as a bioindicator in an urban biotope. The tolerance of these plants to metal toxicity was established in order to determine their possible application in soil phytostabilisation and revegetation in industrial areas that have been contaminated with potentially toxic metals (Serbula et al. 2012 ; Nadgórska-Socha et al. 2013 ; Romeh et al. 2016 ). These results may be useful in evaluating the adaptive properties of these plants to harsh environmental conditions as well as their use in ecological risk assessment (Djingova et al. 2004 ; Przedpełska and Wierzbicka 2007 ; Słomka et al. 2008 ).

The following hypotheses were evaluated:

• Metal pollution contributes to changes in the ecophysiological and morphological properties of selected species within polluted sites compared with plants from a non-contaminated area.
• Plantago species may be useful biological indicators for industrialised urban areas.

Material and methods

The investigated areas represented a variety of habitats (green belts, squares, lawns and park) with ruderal and invasive species such as Robinia pseudoacacia , Solidago canadensis and Reynoutria japonica . Ruderal species were represented by Taraxacum officinale , Achillea millefolium , Bellis perennis , Trifolium repens , Poa annua , Medicago lupulina and others.

The study sites were located in the city of Ruda Śląska (Upper Silesian Industrial District, Southern Poland). For the study, three locations were selected: a road (50°15′17.9″ N, 18°51′17.1″ E), a metallurgical plant (50°17′30.5″ N, 18°52′25.7″ E) and a park (50°16′28.4″ N, 18°50′12.8″ E) (Fig.  1 ).

Location of the study sites in Ruda Śląska: the road (expressway A4); the site of the metallurgical plant “Pokój” and “Strzelnica” Park

The “Strzelnica” Park is a recreational and leisure area and is considered to be a potentially “clean” area. The road is the intersection of the A4 expressway and the provincial road 925, which has intensive road traffic. The metallurgical plant “Pokój,” where steel products are produced and distributed, is a site with a high level of environmental pollution.

Soil and plant material collection

Soil samples were taken from the top layer at 0–10 cm depth from five locations at each site. The samples were collected during the vegetation season in late June and early July 2016. The soil and plant material samples were collected in five replicates at each site (i.e. a total of 15 soil samples and 15 plant material samples).

Plant materials from herbaceous lawns were selected: greater plantain ( Plantago major ) and narrow leaf plantain ( Plantago lanceolata ), which are species of Plantago , family Plantaginaceae . These two ruderal species are common and widespread and are also well known as good biological indicators (Kurteva 2009 , Nadgórska-Socha et al. 2013 , Romeh et al. 2016 , Giacomino et al. 2016 ). The plant material for the biochemical analysis was frozen immediately after collection and kept frozen until the analysis.

Scanning electron microscopy with energy-dispersive X-ray spectroscopy analysis

SEM was used to investigate the micromorphology of the leaf surfaces and stomata size. Leaves from plants of about the same age were taken randomly. Small pieces of fresh leaves near the central nerve (0.5 × 1 cm 2 ) were cut from the same area of the leaf lamina, fixed in 3% glutaraldehyde in a 0.1 M sodium phosphate buffer, washed three times with the same buffer and then dehydrated with ethanol. In the next step, the samples were critical-point dried in a Pelco CPD2 apparatus (Ted Pella Inc., Redding, CA, USA) and then mounted on aluminium stubs with double-sided adhesive carbon tape and at lastly sputter coated in a Pelco SC-6 sputter coater (Ted Pella Inc.) with a 20 nm layer of gold in order to improve the electrical conductivity properties of the samples. All specimens were imaged using a field emission scanning electron microscope (Hitachi SU8010 FESEM; Hitachi High-Technologies Corporation, Tokyo, Japan), which was equipped with a secondary electron detector (ESD). The working conditions were 5 kV or 15 kV accelerating voltages, a working distance (WD) ranging from 8 to 25 mm.

Energy-dispersive X-ray microanalysis (EDX) with a detection limit of 0.1% of weight and beam penetration of 2–5 μm was used to identify the elemental content on the leaf surface using dry plant material that had not been fixed in GA. The parts of the leaves were mounted on aluminium stubs with double-sided adhesive carbon tape and sputter coated with gold. The specimens were examined using a field emission scanning electron microscope (FESEM) and a Thermo Scientific NORAN System 7 energy-dispersive spectrometer (Thermo Fisher Scientific, Madison, WI, USA). Background and element specific peak spectra were analysed with NSS 3 X-ray Microanalysis software (Thermo Fisher Scientific). SEM mode microanalysis was carried out at a 15-kV acceleration and the acquisition time was set to 60 s. Analyses were performed at × 500–× 1100 magnifications on 1–5 points of ten randomly selected pieces of the leaves from all of the investigated sites.

Metal content analysis

The metal content of the soil was determined as pseudo-total HNO 3 extractable fraction as was described in detail by Zheljazkov and Nielsen ( 1996 ). Additionally, metals were also extracted from the soil samples with 0.01 M CaCl 2 (potentially available elements) according to Wójcik et al. ( 2014 ). The metal content was measured in the filtered extracts using atomic absorption spectroscopy (Thermo Fisher Scientific iCE 3500).

Soil pH was determined using a standard method (Ostrowska et al. 1991 ) using a 1:2.5 soil to water ratio. Organic matter content (expressed in %) was estimated following the method of Ostrowska et al. ( 1991 ).

The content of trace elements in the plants was measured using atomic absorption spectrometry (Thermo Fisher Scientific iCE 3500). The plant samples were divided into two groups and analysed as “washed” and “unwashed” samples. The “washed” plants were thoroughly washed with distilled water in an ultrasonic bath (ULTRON, Olsztyn, Poland) for 10 min at 20 °C to remove any dust deposits and then rinsed twice with distilled water. The plant samples were dried at 105 °C and then ground in a stainless steel mill; then, 0.25 g of the samples was wet digested in concentrated HNO 3 at a maximum of 120 °C and finally diluted to 25 ml with deionised water (Lin et al. 2008 ).

Biochemical analyses

Root viability was determined by measuring the GPX activity according to Fang and Kao ( 2000 ). Proline accumulation in the leaves was determined using the acid ninhydrin method (Bates et al. 1973 ). The RWC for the plant samples was determined according to Pathak et al. ( 2011 ). The pH value of the leaves was determined using a pH meter after homogenising 5 g f.w. of the leaves in 10 ml deionised water (Nadgórska-Socha et al. 2017 ). The content of total chlorophyll in the samples was quantitatively determined (Prajapati and Tripathi 2008 ) in accordance with Arnon ( 1949 ). The quantitative determination of ascorbic acid was performed according to Keller and Schwanger ( 1977 ) and as described in detail in Nadgórska-Socha et al. ( 2016 ).

The calculation of the air pollution tolerance index enables the degree of a plant’s tolerance to environmental pollution to be defined. The APTI was calculated according to Prajapati and Tripathi’s ( 2008 ) formula:

where A is the ascorbic acid content (mg g −1 fresh weight); T is the total leaf chlorophyll content (mg g −1 fresh weight); P is the pH of leaf extract; R is the relative water content (%). According to Singh and Rao ( 1983 ), plants with APTI < 10 are sensitive; 10 < APTI < 16 are medium sensitive and APTI > 17 are resistant to air pollution.

Extra material about the methodology that was used is included in the supplementary material (Online Resource 1 ).

Statistical analyses

All of the statistical calculations were performed using Statistica version 13 (StatSoft Inc., Tulsa, OK, USA). The observations were replicated five times for each parameter. The mean standard error was also calculated. Significant statistical differences were estimated using Tukey’s test. The Pearson coefficient of correlation for assessing estimated parameters was also calculated. Analysis of variance (ANOVA) helped to determine the variables that were significantly different among the soil and plant materials.

Results and discussion

Soil analysis.

Soil pollution, particularly due to potentially toxic metal contamination, has been widely investigated by researchers around the world as one of the major environmental problems that can affect plant productivity, the environment and human health (Ross 1994 ; Alloway 1997 ; Kabata-Pendias and Pendias 2001 ; Kandziora-Ciupa et al. 2016 ). Previous soil metal accumulation researches that have been conducted in the urban areas of Upper Silesia (Miasteczko Śląskie, Chorzów, Piekary Śląskie, Sosnowiec, Dąbrowa Górnicza) have also reported excessive concentrations of Pb, Cd and Zn especially in soil samples that had been collected from areas near metallurgical plants (Nadgórska-Socha et al. 2013 , 2016 ; Kandziora-Ciupa et al. 2013 ; Dziubanek et al. 2015 ; Skrynetska et al. 2018 ). Most of these studies were based on the fractions of the extracted elements. According to Zheljazkov et al. ( 2008 ), while the pseudo-total or HNO 3 extractable soil metal concentrations are important, the phyto-available forms of specific metals in the soil are the ones to which plant roots are actually exposed. Amoakwah et al. ( 2013 ) noted that CaCl 2 mobilises both Cd and Zn because of the combined effect of complexation by the chloride anion and cation exchange.

Taking into consideration both points, in our study, we elected to use both methods of metal extraction. According to the Regulation by the Minister of Environment ( 2002 ), the metal concentrations in the soil pseudo-total fraction, particularly cadmium, lead and zinc, exceeded the permissible concentrations at the site of the metallurgical plant site (4 mg kg −1 , 100 mg kg −1 and 300 mg kg −1 , respectively). The potentially toxic elements are usually extracted to a greater extent using HNO 3 extraction rather than CaCl 2 extraction, which was confirmed by our study. In most cases, the potentially bioavailable toxic metal content was below 1% of the estimated content of the elements in the soil fraction that had been extracted using HNO 3 . In the CaCl 2 extracted concentrations, the highest content of Mn, Zn and Cd was recorded at the park, which may be connected with low pH. By contrast, the CaCl 2 extracted fraction had a comparable level of lead with an average of 0.5 mg kg −1 and iron content with an average 0.4 mg kg −1 for all of the investigated sites (Table ​ (Table1). 1 ). Our results are similar to a study in the nearby Miasteczko Śląskie, Poland (Nadgórska-Socha et al. 2016 ). According to Meers et al. ( 2007 ), the 0.01 M CaCl 2 extraction procedure proved to be the most versatile because it provided a good indication of phytoavailability.

Analysis of the soil samples.

Data is expressed as the mean ± SD. The different letters denote significant differences between specific metal concentrations in the fraction that had been extracted with HNO 3 and CaCl 2 , organic matter content, and pH ( p  < 0.05)

The results that were obtained from the investigated locations provide clear information about the impact of pollution on a natural environment that is under pressure from industrialisation and urbanisation. Soils in a city are characterised by a high level of acidity and show a high level of mechanical damage as a result of human activity. Despite this, in our study, the pH of the surface soils at the road and the site of the metallurgical plant were nearly neutral, thus confirming the efficiency of the revitalisation programmes that began in 2015 (The Local Revitalisation Programme of the City of Ruda Śląska until 2030 ( 2015 )). The study of the selected sites showed that the average level of organic matter was 9%. The lowest content was found at the road (Table ​ (Table1 1 ).

Analysis of plant material

Sem observation.

Accumulation of particles on surface of leaves depends on physico-chemical nature of the particulates and the characteristics of the contact surface (Bussotti et al. 1995 ; Liang et al. 2017 ). The interaction between plants and the atmosphere occurs mainly via the stomata and therefore can be considered to be an air quality indicator. A study of the stomatal characteristics is an inexpensive and easy way to obtain relevant results (Kardel et al. 2010 ).

A preliminary examination of the leaves was performed using light microscopy. Plantago major leaves have a blunt apex, 3–9 nerves, are sometimes slightly serrated, a naked or slightly hairy surface and a round shape. The leaves of Plantago lanceolata have a lanceolata or elliptic shape. Its leaf blade is usually full and rarely has a few serrations. In both of the investigated species, the abaxial surface of the leaves is lighter than the adaxial surface. No epicuticular waxes were present on the surfaces of the leaves. Fine deposits with irregular shapes and of different sizes were seen on the surfaces of the leaves in a polluted environment (the area near the road and the site of the metallurgical plant) (Fig.  2b, c, e, and f ). At the road, the stomata were mostly closed and blocked by dust (Fig. 2b, e ). Single trichomes were rarely observed on surfaces of the leaves from all of the investigated sites (Fig. 2b, c, and f ).

Representative SEM images of the adaxial surfaces of the leaves. a Plantago lanceolata at the park. b Plantago lanceolata at the road. c Plantago lanceolata at the site of the metallurgical plant. d Plantago major at the park. e Plantago major at the road. f Plantago major at the site of the metallurgical plant

Amphistomatous leaves and stomata occurred on both sides of the leaves in all of the Plantago lanceolata and Plantago major plants that were observed. The study of the micromorphology and anatomy of the Plantago lanceolata leaves using SEM revealed differences in the SPL in the area that has heavy traffic and near the site of the metallurgical plant compared with the park. The highest SPL values were found in the Plantago lanceolata (24.04 ± 1.26 μm) leaves at the park. Despite the fact that the SPL values in the leaves of Plantago major were much lower at the park (16.5 ± 0.75 μm), the lowest values for Plantago lanceolata (13.58 ± 0.95 μm) and for Plantago major (14.53 ± 0.65 μm) were recorded at the road. At the site of the metallurgical plant, the average SPL was 16.4 ± 0.91 μm and 18.23 ± 0.6 μm for Plantago lanceolata and Plantago major , respectively. The leaves of Plantago major had a comparable SPL at all of the investigated sites. A strong positive correlation was observed between the SPL and RWC, total chlorophyll content and APTI ( r 2  = 0.7, r 2  = 0.55 and r 2  = 0.7, respectively) and a negative correlation was observed between the SPL and ascorbic acid and proline content ( r 2  = − 0.58 and r 2  = − 0.81, respectively). No correlation was observed between the SPL and metal content in either the washed or unwashed plant samples.

Wagoner ( 1975 ) reported no differences in the size of the stomata between polluted and unpolluted sites. Alves et al. ( 2008 ) described that an increase in stomatal density together with a decrease in stomatal size leads to an optimal adjustment for the control of gas exchange and the entrance of pollutants through the stomata. Moreover, Kardel et al. ( 2010 ) noticed a decrease in both the adaxial and abaxial stomata sizes in the leaves of Plantago lanceolata that acts as a mechanism for adapting to pollution stress in unsuitable habitats. The formation of smaller stomata in the leaf epidermis of trees was also recorded in Lublin, Poland (Chwil et al. 2015 ).

X-ray microanalysis

Quantitative EDX analysis only provided information on the distribution of the elements and was not sensitive in depicting low concentrations of the elements (below the detection limit (> 0.1% weight)). We also analysed the elemental composition of the particles on the adaxial leaf surfaces. X-ray microanalysis revealed the presence of Si, Fe, S, Na, Ca, Mg, Cl, O, K and Al over the entire leaf surface sections that were examined. The results are presented as the averages of the spectra that were obtained at the study sites (Fig.  3 ). The gold (Au) signals can be considered to have originated from the sputter coating.

Selected SEM-EDX images of Plantago leaf samples with the chemical compositions of the most frequently identified elements on the adaxial surfaces. a , b The park. c , d The road. e , f The site of the metallurgical plant

The element content on the adaxial leaf surfaces from the park was mainly represented by O, C, K, Cl, Ca and Mg (Fig. 3a, b ), while at the road and the site of the metallurgical plant, additional elements were present (Na, Al, Si and Fe) (Fig. 3c–f ). The highest peaks of Si and Al were recorded at the road, and a higher Fe content was recorded at the site of the metallurgical plant, along with the presence of Mn and fungal spores (not shown).

Almost the same elemental content was recorded on the surfaces in Bignoniaceae family leaves that had been collected from different areas of the Pune D istrict, India (Kedar et al. 2018 ). In research conducted by Weerakkody et al. ( 2018 ) near a busy road, the amounts of C and O, in addition to Fe and Cl, were considerably larger compared with the other elements in PM 10 , and Ca, K, Si, Mg and S were present in particles of various sizes distributed on the leaves of all investigated species. According to Weerakkody et al. ( 2018 ), a high content of C and O can also indicate the presence of carcinogenic polycyclic aromatic hydrocarbons, primarily from fuel exhausts and tyre wear. Trace amounts of Ca, Ba, Mn, K, Mg and Zn can also be present in vehicle exhausts bound to organic components (Lin et al. 2005 ; Sharma et al. 2013 ). In addition to the dust that originates from road traffic, PM 10 containing Al, Ca, Na, Si, Cl, F and N can originate from soil dust (Maher et al. 2013 ; Weerakkody et al. 2018 ).

Analysis of the metal content in the plant material

According to other researchers, the foliar metal uptake is mainly due to the soil–root pathway in urban and industrial environments (Schreck et al. 2012 ; Dao et al. 2014 ; Kandziora-Ciupa et al. 2017 ). The concentrations of the elements (Fe, Mn, Pb, Cd, Zn) were investigated in both washed and unwashed leaves of Plantago major and Plantago lanceolata. Higher metal concentrations were found in the unwashed samples, which was predictable as the dust on the surfaces of leaves can also contain metals (Maher et al. 2013 ; Weerakkody et al. 2018 ). In the washed plant material, the highest accumulation of Mn, Fe and Pb was recorded in the leaves of Plantago lanceolata at the site of the metallurgical plant (Table ​ (Table2 2 ).

Analysis of the metal content in Plantago lanceolata (Pl) and Plantago major (Pm)

Data are expressed as the mean ± SD. The different letters denote significant differences between the metal concentrations in the various plants in the washed and unwashed samples ( p  < 0.05).

In the study area, the highest concentrations of Zn were found in the unwashed samples at the site of the metallurgical plant site for both of the species that were studied. The average was 61 mg kg −1 for Plantago major and 43 mg kg −1 for Plantago lanceolata in the washed samples, and the average was 106 mg kg −1 for Plantago major and 88 mg kg −1 for Plantago lanceolata in the unwashed samples (Table ​ (Table2 2 ).

The iron content in the study area ranged from 51 to 391 mg kg −1 for the washed samples and from 308 to 2830 mg kg −1 for the unwashed samples. The highest concentrations were observed in both study species at the site of the metallurgical plant. The Pb concentration was a few times greater in the unwashed than in the washed plants. The highest manganese concentration was recorded in the leaves of Plantago major in the unwashed samples (792 mg kg −1 ) at the site of the metallurgical plant, which is eight times higher than the concentration in the washed samples (Table ​ (Table2 2 ).

Potentially toxic concentrations of Cd (> 5 mg kg −1 ) were found in all of the samples of Plantago lanceolata leaves from the entire study area, according to the limits reported by Kabata-Pendias and Pendias ( 2001 ). Moreover, the permissible Cd content was exceeded in almost all of the unwashed samples, except for the leaves of Plantago major at the road (Table ​ (Table2). 2 ). Cd accumulation in edible plants has been found at significantly lower concentrations, i.e. 0.8 to 0.1 mg kg −1 (Dziubanek et al. 2015 ) compared with our results. A field study conducted by Nadgórska-Socha et al. ( 2017 ) also reported a lower metal content in Taraxacum officinale , Plantago lanceolata , Betula pendula and Robinia pseudoacacia leaves. Stafford et al. ( 2016 ) noted that the Cd accumulation in Plantago lanceolata ranged from 0.44 to 0.89 mg kg −1 . In our study, the highest concentration of Zn in the washed samples, which exceeded the permissible concentration 100 mg kg −1 (Kabata-Pendias and Pendias 2001 ), was found in the leaves of Plantago major at the park site. By contrast, the study conducted by Kurteva ( 2009 ) recorded a higher Zn accumulation in the leaves of Plantago lanceolata. Our investigation of the accumulation of potentially toxic metals found much higher metal concentrations in the soil and leaves of Plantago major compared with the study in Cluj-Napoca, Romania, of Levei et al. ( 2018 ).

After averaging the data that was obtained in the measurements, the concentrations of the elements in the washed samples can be ranked in the following descending order for Plantago lanceolata : Fe > Mn > Zn > Cd > Pb and for Plantago major : Fe > Zn > Mn > Pb > Cd as was also recorded for Plantago major in Sosnowiec (Skrynetska et al. 2018 ). The order of the concentrations in the unwashed samples for both Plantago species was the same: Fe > Mn > Zn > Pb > Cd. This fact supports the crucial point that the plant samples that are used in biomonitoring studies must be washed.

Analysis of the biochemical parameters

To estimate the state of the environment, in addition to the above-mentioned results, the total chlorophyll, proline, ascorbic acid, relative water content and leaf pH were determined. The GPX activity was also analysed (Table ​ (Table3 3 ).

Analysis of the biochemical parameters of Plantago lanceolata (Pl) and Plantago major (Pm)

Data are expressed as the mean ± SD. The different letters denote significant differences between the contents of AA, Chl, RWC, pH, GPX, and proline ( p  < 0.05)

Proline accumulation is considered to be a common physiological response of many plants to environmental stress factors (Verbruggen and Hermans 2008 ; Tantrey and Agnihotri 2010 ). Moreover, researchers have found a significant amount of proline in the reproductive parts of different plant species, which raises the possibility that the accumulation of this amino acid may also occur in non-stressed physiological conditions (Mattioli et al. 2009 ). Numerous studies have also noted a higher content of proline in samples from contaminated areas compared with potentially clean sites (Tantrey and Agnihotri 2010 ; Kumar et al. 2010 ; Kandziora-Ciupa et al. 2016 ; Kandziora-Ciupa et al. 2017 ). In our investigation, the highest proline content was recorded at the road site for both of the study species (Table ​ (Table3). 3 ). The average proline contents for the Plantago major and Plantago lanceolata leaves were 7.8 μmol g −1 and 8.5 μmol g −1 f.w., respectively (Table ​ (Table3). 3 ). An increase in the proline level during environmental contamination was also found in Philadelphus coronarius leaves by Kafel et al. ( 2010 ) and confirmed in Taraxacum officinale , Plantago lanceolata , Betula pendula and Robinia pseudoacacia leaves because of urban environmental traffic contamination by Nadgórska-Socha et al. ( 2017 ). In a field study near the site of a smelter, a higher proline content was also recorded in the leaves of Vaccinium murtillus (Kandziora-Ciupa et al. 2017 ).

GPX activity is significant for plant growth and development. The activity of antioxidant enzymes changes under biotic and abiotic stress conditions and can be used as a potential indicator of metal toxicity and other stress factors (Verma and Dubey 2003 ; Doğanlar and Atmaca 2011 ; Kandziora-Ciupa et al. 2017 ). According to the obtained results, a higher level of GPX activity was recorded in the leaves of Plantago major at the site of the metallurgical plant (1254 tetra-guaiacol g −1  f.w.), while the lowest was recorded in the Plantago lanceolata leaves (348 tetra-guaiacol g −1  f.w.) at the road (Table ​ (Table3). 3 ). In our study, a lower GPX activity was recorded at the road and was positively correlated with the Fe and Mn content in the washed samples and with Pb, Zn, Fe and Mg in the unwashed samples. A similar dependence was also found in studies that were conducted by Kandziora-Ciupa et al. ( 2013 , 2017 ). Many authors have reported increased GPX activity in response to elevated potentially toxic metal concentrations (Verma and Dubey 2003 ; Kafel et al. 2010 ; Doğanlar and Atmaca 2011 ; Nadgórska-Socha et al. 2013 ; Marchand et al. 2016 ).

Relative water content (RWC) is the level of water that is required in plants to maintain a physiological balance (Rai and Panda 2014 ). According to Krishnaveni ( 2013 ), RWC is one of ecophysiological indicators of environmental stress in plants. In our study, the average relative water content in the leaves of Plantago major was nearly 64% and was nearly 67% for Plantago lanceolata , which confirms that the selected plants are resistant to water stress. The lowest values for both species were observed at the road (Table ​ (Table3 3 ).

The leaf pH, which is a common physiological parameter, is also suggested to be an indicator of plant stress (Krishnaveni et al. 2013 ; Husson et al. 2018 ). The pH of the extracts from the leaves in the study area ranged from 4.5 to 5.6. The lowest pH was recorded in both study species at the road. The average pH for the Plantago major leaves was 5.21, and it was 4.98 for Plantago lanceolata (Table ​ (Table3). 3 ). The values of the leaf pH that were obtained were lower for both species than the results of a field study in Sosnowiec (Poland) (Skrynetska et al. 2018 ). Krishnaveni et al. ( 2013 ) also recorded a decrease in the leaf pH values at polluted sites. By contrast, a laboratory study conducted by Cornelissen et al. ( 2011 ) reported that leaf pH is largely a species-specific trait, and therefore, the investigated species could maintain a leaf pH independently from the soil environment. Studies conducted by Sharma et al. ( 2013 ), Zhang et al. ( 2016 ) and Bharti et al. ( 2018 ) emphasised that a lower leaf pH is connected with the presence of SO x and NO x in the air. This fact suggested us to conclude that the leaf pH depends directly on air quality.

Studies on chlorophyll content are considered to be relevant as its level is connected with tolerance in contaminated environments (Pathak et al. 2011 ; Rai and Panda 2014 ; Ogunkunle et al. 2015 ; Nadgórska-Socha et al. 2017 ). We observed comparable results for Plantago major and Plantago lanceolata . The average contents in the leaves of Plantago major and Plantago lanceolata were 0.14 mg g −1 f.w. and 0.13 mg g −1 f.w., respectively (Table ​ (Table3). 3 ). Previous field studies have recorded a higher total chlorophyll content in the leaves of Plantago major and Plantago lanceolata in Sosnowiec, Poland (Skrynetska et al. 2018 ) and in the leaves of Plantago lanceolata in Dąbrowa Górnicza, Poland (Nadgórska-Socha et al. 2017 ) . The content of Chl can be affected by high temperature, drought, salt stress, light intensity, gaseous pollutants and potentially toxic metal contamination (Pandey et al. 2015 ; Zhang et al. 2016 ).

Another important indicator of physiological condition of a plant is the content of ascorbic acid (AA), which is a strong reducing agent that activates many defence mechanisms in plants, whereby increased ascorbic acid content enhances pollution tolerance (Pandey et al. 2015 ; Zhang et al. 2016 ; Nadgórska-Socha et al. 2017 ). Ascorbic acid is located mainly in the chloroplast and plays an important role in the synthesis of the cell walls, cell division and the processes that are associated with detoxification (Ogunkunle et al. 2015 ). In our study, the average AA content in the leaves of Plantago major was 0.46 mg g −1  f.w., while for Plantago lanceolata , it was 0.37 mg g −1 f.w. The lowest AA content was observed in the leaves of Plantago lanceolate at the park (Table ​ (Table3). 3 ). A much lower AA content was found in the leaves of Plantago major in a field study in Sosnowiec, Poland (Skrynetska et al. 2018 ). As was reported by Tripathi and Gautam ( 2007 ), an increase in the AA content in all plant species may be due to the increased rate of the production of reactive oxygen species. In a field study conducted by Nadgórska-Socha et al. ( 2016 ), a decreasing tendency was found in the leaves of R. pseudoacacia , and an increase in the AA content was found in the leaves of M. album at contaminated sites. Some studies have also reported a high concentration of AA at industrial sites (Agbaire and Esiefarienrhe 2009 ; Meerabai et al. 2012 ; Ogunkunle et al. 2015 ).

Calculating the air pollution tolerance index (APTI) enables the tolerance of plants to air pollution to be determined and the biochemical parameters that are responsible for resistance to environmental stress factors to be found. In our study, Plantago major and Plantago lanceolata had a narrow range of tolerance in the APTI index (5.6 to 7.4). It was found that the relative APTI average of Plantago major was 6.7 while it was 6.8 for Plantago lanceolata , thus indicating that both are sensitive to air pollution. The road site had the lowest APTI values for both of the study species (Table ​ (Table3). 3 ). According to the classification of Singh and Rao ( 1983 ), the investigated plants are species that are sensitive to air pollution. Low values of APTI were also noted in both contaminated and conventionally clean sites in Sosnowiec, Poland (Skrynetska et al. 2018 ). In Dąbrowa Górnicza, Poland, the APTI of Plantago lanceolata was higher (8.43–14.57), especially at non-contaminated sites compared with contaminated sites (Nadgórska-Socha et al. 2017 ). Another study in Southern Poland (Miasteczko Śląskie, Katowice, Jaworzno) using Robinia pseudoacacia and Melandrium album at potentially toxic metal-contaminated sites recorded a mean APTI value for all of the investigated sites at 9.4 for R. pseudoacacia and 8.7 for M. album (Nadgórska-Socha et al. 2016 ). Zhang et al. ( 2016 ) identified species that are tolerant to air pollution ( Magnolia denudata , Diospyros kaki , Ailanthus altissima , Fraxinus chinensis and Rosa chinensis ), which had been collected from two heavy traffic roadside sites and one suburban site in Beijing and recommended it to be planted at locations where there is heavy traffic. Bharti et al. 2018 estimated the APTI of 25 plant species that were growing at the Talkatora Industrial Area, India, and determined that Polythalia longifolia was the species that was most sensitive to air pollution.

A plant species with a higher APTI can be used in green belts and should be given priority for replantation in urban and industrial areas in order to reduce the effects of air pollution (Sisodia and Dutta 2016 ; Achakzai et al. 2017 ; Bharti et al. 2018 ). A plant species with a lower APTI can be recommended as a bioindicator and for environmental monitoring (Nadgórska-Socha et al. 2016 , 2017 ; Bharti et al. 2018 ). The results that were obtained indicate that Plantago major and Plantago lanceolata species can be classified as being sensitive to air pollution and can be recommended for bioindicative research in urban and industrial areas.

A clear correlation was found between the pH value and the content of Mn and Zn ( r 2  = 0.5 and r 2  = 0.87, respectively) and between the Chl and Pb content ( r 2  = − 0.85) in the washed plant material. Much stronger correlations were observed in the unwashed material. A correlation was found between the dehydrogenase activity and the content of Pb, Fe, Mn and Zn ( r 2  = 0.76; r 2  = 0.92; r 2  = 0.96 and r 2  = 0.57, respectively). Significant positive correlations were found between the RWC and Cd and Zn concentrations ( r 2  = 0.81; r 2  = 0.7, respectively), between the total chlorophyll and Cd content ( r 2  = 0.66) and between the pH value and the content of Zn ( r 2  = 0.72). Negative correlations were observed between proline content and the content of Pb, Fe, Zn and Cd in the unwashed samples ( r 2  = − 0.61; r 2  = − 0.76; r 2  = − 0.58 and r 2  = − 0.56, respectively).

Although the Plantago major and Plantago lanceolata species that were investigated demonstrated different ecophysiological responses to environmental pollution, they can be recommended as unified bioindicators because of their wide dispersion in Europe, North America, and other regions of the world, e.g. South Africa (Kardel et al. 2010 ). The ability of this plant to accumulate metals can be also used in phytostabilisation and environmental risk assessment studies (Gucwa-Przepióra et al. 2016 ; Romeh et al. 2016 ). Moreover, it is important to continue this kind of research in order to determine plants with a tolerance or resistance to environmental pollution that can be used in developing green belts or to provide a low-cost and eco-friendly approach for reducing air pollution.

Conclusions

The examinations of the leaves of Plantago major and Plantago lanceolata showed anatomical, biochemical and ecophysiological changes in the plant samples that had been collected from an industrialized urban area. Strong correlations were found between the SPL and the ecophysiological parameters (RWC, APTI, Chl, AA, proline content). The metal content also correlated with the biochemical and ecophysiological indexes to different degrees depending on the specific element.

The difference in metal concentrations between the washed and unwashed plant material is an essential distinction. The statistical analysis demonstrated the necessity of washing the plant material that is used in metal bioaccumulation studies because this factor affects the experimental accuracy.

According to the SEM-EDX results, a higher content of Al, Fe, Si and Mn was observed on the adaxial leaf surfaces at the road and metallurgical plant sites. During such an analysis, researchers should take into account the detection limit and depth of the beam penetration because tracking the trace element content in particles >2 μm is quite difficult. Therefore, for bioaccumulation studies, SEM-EDX analysis with an additional analysis of the metal concentration (AAS, ICP etc.) is recommended.

The results demonstrated that Plantago major had a higher tolerance ability to environmental pollution compared with Plantago lanceolata at the site of the metallurgical plant – an area with an extremely high metal content, which ensured its greater adaptation ability to stress factors . The calculation of the APTI index demonstrated that the plant species that were studied have a narrow range (5.6–7.4) and are sensitive to air pollutants, including potentially toxic metals, which suggests their usefulness as bioindicators of the environmental state.

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Acknowledgements

We would like to express our gratitude to the anonymous reviewers for their careful reading of our manuscript and their insightful comments and suggestions.

Abbreviations

This work was co-funded by the university statutory activity funds for young researchers.

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The authors declare that they have no conflict of interest.

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