Environmental and Experimental Botany 147 (2018) 31–42

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

How does the endophytic fungus Mucor sp. improve Arabidopsis arenosa T
vegetation in the degraded environment of a mine dump?
⁎
P. Rozpądeka, , A. Domkab, R. Ważnya, M. Nosekc, R. Jędrzejczyka, K. Tokarzd, K. Turnaub
a
Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387 Kraków, Poland
b
Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
c
Institute of Biology, Pedagogical University, Podchorążych 2, 30-084 Kraków, Poland
d
Institute of Plant Biology and Biotechnology, University of Agriculture, Aleja 29 Listopada 54, 31-425 Kraków, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: The endophytic fungus Mucor sp. was isolated from Arabidopsis arenosa inhabiting post mining wastes lands. Its
Auxin role in plant adaptation to toxic metal enriched environment was evaluated. Plants inoculated with the fungus
Endophytic fungi yielded significantly more biomass. Their growth response was correlated with significant elongation of root
Ethylene hairs, an improved water and P status and a significant upregulation of the expression of genes associated with
Nutrients
nutrient uptake. The mechanism of root hair elongation was investigated with auxin and ethylene insensitive
Phosphorus
Arabidopsis thaliana mutants. The results clearly indicate that the root hair elongation phenotype results from
Root hairs
Toxic metals fungi induced alterations in ethylene metabolism. The upregulation of close to 50 genes associated with ethylene
biosynthesis and signaling confirm these results. The accumulation of Zn and Fe was lower in endophyte in-
oculated plants. Additionally, root to shoot translocation of Fe, Cd and Zn was improved. The expression of metal
transporters associated with metal influx, efflux and distribution within the plant corresponded with altered
metal homeostasis. The results of this study clearly show that the endophytic fungus plays an important role in
the adaptation of the non-mycorrhizal A. arenosa to metal toxicity.

1. Introduction severe risks for human health and agriculture. In degraded site man-
agement plants play an essential, remediating role, thus several efforts
There is a growing number of evidence indicating the importance of have been made to improve plant toxic metal tolerance (Ali et al., 2013;
endophytic fungi in plant growth, fitness and adaptation to the en- Rozpądek et al., 2017).
vironment. According to the definition, fungal endosymbionts are a An important but underestimated aspect of toxic metal tolerance is
diverse group of microorganisms inhabiting plant tissues without the ability of the plant to form mutual associations with symbiotic
causing any visible symptoms of disease (Rodriguez et al., 2009; Schulz fungi. The best described and probably most common beneficial plant-
and Boyle, 2005). Their existence, ubiquity and abundance have been fungi symbiosis is the association with mycorrhizal fungi (Martin and
long recognized, however, only recently, their ability to accelerate Kohler, 2013). The role of mycorrhiza in toxic metal stress tolerance
plant growth and to protect plants against a wide variety of stress and the mechanisms of mycorrhiza dependent trace metal management
factors has gained the attention of the scientific community (Van Der have been previously described. Numerous reports indicate that my-
Heijden et al., 2008; Yuan et al., 2010; Kanchiswamy et al., 2015; corrhiza greatly improves plant fitness in metalliferous soils (Orłowska
Kauppinen et al., 2016; Rho et al., 2017). et al., 2012; Rozpądek et al., 2014; Ruytinx et al., 2016; Turnau et al.,
Metal pollution has dramatically increased during the last centuries 2010). Not all plants, however, have the ability to form functional
and it is expected to rise in the future. As a result, further losses in the symbiosis with mycorrhizal fungi, including numerous metallophytes
complexity and bio-diversity of environments are expected. and hyperaccumulators from the Brassicaceae family. Species from this
Contamination of the food chain due to the transfer of toxic metals from family are one of the most highly represented plants inhabiting en-
the soil to plants and to ground and drinking water reservoirs cause vironments enriched in toxic metals including degraded, post-industrial

Abbreviations: ABA, abscisic acid; ABC, ATP-ase binding cassette; ET, ethylene; EtOH, ethanol; BR, brassinosteroids; CDF, cation diffusion facilitator; JA, jasmonic acid; MS, Murashige
and Skoog; MSR, Strullu-Romand medium; NaOCl, sodium hypochlorite; NRAMP, natural resistance-associated macrophage protein; PGPR, plant growth promoting rhizobacteria; SA,
salicylic acid; SL, strigolactones; ZIF, zinc induced facilitator; ZIP, zinc/iron-like protein
⁎
Corresponding author.
E-mail address: piotr.rozpadek@uj.edu.pl (P. Rozpądek).

https://doi.org/10.1016/j.envexpbot.2017.11.009
Received 23 August 2017; Received in revised form 17 November 2017; Accepted 18 November 2017
Available online 21 November 2017
0098-8472/ © 2017 Published by Elsevier B.V.
P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

Table 1
Element concentration in substratum from the “Bolesław” mine dump.

Element Zn Pb Cd Fe K P/Plabile
Concentration

“Bolesław” 12680 ± 380 5240 ± 160 79.0 ± 2,4 (5.83 ± 0,43) 104 22 ± 15 1200 ± 300/12.0 ± 6.4
[mg kg−1]
Aro [mg kg−1] 156 ± 33 – – (7.5 ± 1.8) 103 1750 ± 38 4900 ± 1500/78.0 ± 6.7
Literature averages 3.0–2900, median 52 17 (Steinnes, 0.1–1 (Smolders (2–4 104) (Cornell and (0.4 –30) 103 502/30.5 (Deng et al., 2017)
[mg kg−1] (Mertens and Smolders, 2013) and Mertens, 2013) Schwertmann, 2003) (Zörb et al., 2014)
2013)

habitats (van der Ent et al., 2013; Verbruggen et al., 2009). The role of the root, the plant deals with it either by storing it in vacuoles of root
fungal symbionts in Brassicaceae plants metal management has not been cells or by transporting it radially through the root to be loaded into the
described yet. xylem for transport to the shoot (Milner and Kochian, 2008). Metal
Several reports indicate that Arabidopsis thaliana can benefit from uptake and distribution is regulated by a complex network of metal
symbiosis with root endophytes, basidiomycete Piriformospora indica carriers. Protein transporters from various families such as the CDF
and Sebacina vermifera (Lahrmann et al., 2015; Verma et al., 1998) as (cation diffusion facilitator), NRAMP (natural resistance-associated
well as species from the Trichoderma genus (Brotman et al., 2013; macrophage protein), ABC (ATP-ase binding cassette), ZIP (zinc/iron-
Contreras-Cornejo et al., 2009). To this date reports indicating im- like protein) and ZIF (zinc induced facilitator) were shown to play a
proved growth or fitness, drought stress and induced resistance against significant role in trace metal homeostasis maintenance (Mirzahossini
pathogens have been published (Oelmüller et al., 2009; Stein et al., et al., 2015). A tightly regulated mechanism of adapting carrier quan-
2008; Waller et al., 2005). Recently, A. thaliana was found to harbor a tity/exposition to current metal availability and thus, controlling metal
number of fungal endophytes, in both shoots and roots, however, their flux from the rhizosphere into the plant and distribution within its
ecological significance is not fully recognized (García et al., 2013; tissues determines metal tolerance and supply (DalCorso et al., 2013;
Junker et al., 2012). According, to a most recent report, the A. thaliana Verbruggen et al., 2009).
root endophyte Colletotrichum tofieldiae improved plant growth and The aim of this study was to investigate the role of Mucor sp. in
fitness by improving P solubilization and uptake from the substratum in adaptation of Arabidopsis arenosa to a highly polluted environment of a
P limiting conditions (Hiruma et al., 2016). The role of endophytes in mining waste dump. Mucor sp. is a member of the Mucormycetes, re-
facilitating vegetation (species other than Arabidopsis) in environments cently shown to associate with extant, basal land plants, such as liver-
highly polluted with toxic metals has also been indicated (Li et al., worts, hornworts and lycopods, in a symbiosis whose mutualistic nature
2011; Wężowicz et al., 2017; Deng et al., 2011), however, our knowl- is suspected (Plett and Martin, 2015). The fungus was isolated from
edge of the mechanisms enabling plant-endophyte consortia to thrive plants inhabiting the “Bolesław” Zn, Cd and Pb mine dump, located in S
under such conditions is scarce. Poland (50°16′58″N 19°32′9″E), in close proximity to the “Śląsk/Si-
Plants exposed to stress exhibit a broad range of morphogenic lesia” mining region. A detailed description of the climatic and phy-
changes. Adaptations in root architecture and activation of root hair sical-chemical properties of the soil can be found in Orłowska et al.
elongation are probably the best described adaptations to suboptimal (2005).
soil conditions including environments enriched in toxic metals or/and
low nutrient availability (Keunen et al., 2016; Potters et al., 2007). Root 2. Materials and methods
hairs are tubular epidermal outgrowths which account for up to 91% of
total root surface area (Gahoonia and Nielsen, 1998; Bates and Lynch 2.1. Plant cultivation
1996), but more importantly root hairs are responsible for the majority
of water and nutrient uptake, particularly when exposed to nutrient Following harvest plants were cultivated in pot culture in a green
deficiencies (Gahoonia and Nielsen, 1998; Grierson and Schiefelbein, house in commercial soil (ARO, PL for description see Table 1) to obtain
2002). Another important role of root hairs is modifying the rhizo- seeds. Seeds were surface sterilized with 8% NaOCl for 5 min, 96%
sphere by exudation of organic acids, enzymes, chelating agents and EtOH and 75% EtOH for 1 min, rinsed in distilled water 3 times, sown
other secondary metabolites (Grierson and Schiefelbein, 2002). to petri dishes with 1/4 MS medium and placed in darkness for 48 h.
Root hair growth is modulated by the action of plant hormones: Subsequently, seeds were transferred to a growth chamber (Panasonic
ethylene (ET), auxin, strigolactones (SL), jasmonic acid (JA) and bras- MLR-352H-PE, JPN) with a 16 h photoperiod, 21/17 °C day/night
sinosteroids (BR) (Koltai and Kapulnik, 2011; Lee and Cho, 2013; temperature and 50% humidity. After 10 days seedlings were moved to
Muday et al., 2012; Zhu et al., 2006). The best described and probably MSR medium with no sugar (10 plants per petri dish) to facilitate fungi
most significant role play ET and auxin. In regard to root hair growth colonization and inoculated with the fungus. Inoculation was per-
the functional relationship between ET and auxin is synergistic, both formed by placing 2 × 2 mm pieces of mycelium in 5 mm distance from
hormones play a positive role in this phenomenon (Muday et al., 2012). the root tip. Inoculated plants were described E+, not inoculated E-.
In addition to adaptation to abiotic factors, root hair elongation (and After 10 days of growth, plants were transferred to 80 ml multipots (1
plant growth acceleration in general) was shown to be induced by PGPR plant per pot) filled with sterile substratum from the mine dump ‘Bo-
(plant growth promoting rhizobacteria) via altering the homeostasis of lesław’ mixed with sterile sand in a 2:1 (v/v) ratio or a mixture of ARO
these two hormones (Poupin et al., 2016). Arabidopsis growth and root soil with sand in a 2:1 (v/v) ratio for control. The substrate from the
architecture were also improved by inoculation with Trichoderma virens mine dump contained elevated quantities of Pb, Zn and Cd, the abun-
and T. atroviride. The response was suggested to be associated with dance of essential macronutrients (N, P, K) was very low (Table 1).
stimulating auxin signaling/auxin production by the fungus (Contreras- Multipots were placed in 23 × 45 × 18 cm plexiglass chambers (45
Cornejo et al., 2009). pots per chamber) covered with polyamide gauze to avoid contamina-
Effective trace metal (Cu, Fe, Zn) management (uptake, compart- tion. Plants were grown in the growth chamber for 25 days. Alter-
mentation and detoxification) is required for sufficient supply to dif- natively, 5-day old seedlings of A. arenosa were inoculated with Mucor
ferent plant organs and in prevention of nonessential metals (Cd, Co, sp. (NCBI accession number KU234656) by adding 3.2 × 105 spores (in
Pb) from inducing deleterious effects on plant cells. Once metals enter suspension) to a single pot. Three independent chambers per treatment

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P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

were prepared with a total of 115 control and 132 inoculated plants. (Cy3) target RNA to generate cRNA for oligo-microarrays. Gene
Expression Hybridization Kit was used for fragmentation and hy-
2.2. Fungi visualization bridization and Gene Expression Wash Buffer Kit was used for washing
slides after hybridization. Acquisition and analysis of hybridization
For optical microscopy Arabidopsis tissues were stained with aniline intensities were performed using Agilent DNA Microarray Scanner
blue and Sudan IV according to (Atsatt and Whiteside, 2014). At least G2505C. After microarray scanning, data were extracted, and back-
ten seedlings were analyzed. ground subtracted using the standard procedures from the Agilent
Feature Extraction (FE) Software version.
2.3. CO2 assimilation and transpiration

Gas Exchange measurements were carried out on 3-week-old A. 2.8. Data processing and statistical analysis
arenosa rosettes with a portable open gas exchange system (Li-6400, Li-
Cor, Lincoln NE, US) equipped with a 6 cm2 cuvette and 6400-02B LED Data preprocessing was performed by Genespring 14.8 software
light source. Before data recording, rosettes were placed in the cuvette (Agilent Technologies, USA). Input expression values were normalized
for 2 min to allow photosynthesis to stabilize. Measurements were by the percentile shift normalization algorithm (75th percentile) of
performed in CO2 saturated conditions (650 μmol mol−1); logarithm transformed data. Differential gene expression was evaluated
300 μmol s−1 of air flow, 50–55% relative humidity, 25 °C leaf tem- by comparing gene expression profiles of plants grown in control sub-
perature and under the 130 μmol (quantum) m−2 s−1 red light intensity strate, toxic metal enriched substrate (TM) and TM inoculated with
in ten biological replicates (10 plants). Mucor sp. Statistical significance was assessed with the Student’s t-test,
with a 2-fold up or down regulation cut-off and corrected p-value cut-
2.4. Phytohormone concentration determination off 0.05. Multiple testing correction was performed by Benjamini
Hochberg FDR. Global gene expression was quantified from two in-
Unlabeled ABA, JA, SA, were purchased from Sigma-Aldrich (D). dependent samples (three plants per sample) per treatment. Microarray
Sample preparation and HPLC analysis were carried out according to data was deposited in the NCBI database (http://www.ncbi.nlm.nih.
(Müller et al., 2011) with modifications. Frozen plant roots (about gov/geo) under the accession number GSE100525.
200 mg FW) was powdered in liquid nitrogen with a metal pestle in
polypropylene tubes and then extracted with methanol:isopropa- 2.9. qPCR
nol:glacial acid (20:79:1% v/v/v) in a 10:1 v/w ratio for 20 min in 4 °C.
During extraction sonication was applied. Subsequently, samples were Reverse transcription was carried out on 1000 ng of total RNA, after
centrifuged for 20 min in 15,000g. This procedure was performed 5 digestion with DNase (DNA free kit, Ambion Bioscience, US), with
times to assure maximum, close to 100% extraction (from the second iScript cDNA synthesis kit (Bio-Rad, US). For qPCR, probes were labeled
extraction 1 ml of extraction solution was used). Three plants were with the EVAGreen (SsoFast EvaGreen Supermix, Bio-Rad, US) fluor-
pooled together for a single sample. Hormones were extracted from five escent dye. For a single reaction 10 ng of cDNA and 150 nM of gene
samples per treatment. specific primers were used. To test amplification specificity a dis-
sociation curve was acquired by heating samples from 60 °C to 95 °C. As
2.5. HPLC analysis house-keeping reference α-tubulin 5 was used. Reaction efficiency was
tested by serial dilutions of cDNAs with gene specific primers
The HPLC analysis was performed using Shimadzu LCMS-2020 (Supplementary data). All samples were run in triplicates. Expression
(JPN) system equipped with an autosampler. Separation of plant ex- was calculated according to Pfaffl (2001). with plants grown in sub-
tracts was performed with a Kinetex 2,6 u C18 100 × 2.1 mm column. stratum lacking toxic metals (control plants) serving as calibrator. Gene
The total eluent flow was 0.400 ml min−1. Gradient profile described in expression was quantified from five samples per treatment (three plants
(Müller et al., 2011). The MS analysis was performed using quadrupole per sample).
mass spectrometer (Shimadzu) negative modes for JA, SA, ABA. The
following MS parameters were used for analysis: DL temp: 250 °C, HB
temp: 200 °C, detector voltage: 0,95 kV, oven temp: 35 °C, nebulizing 2.10. Phosphorus and toxic metal concentration analysis
gas flow: 15 l min−1. The external standard calibration curve method
was used for determination of hormones content in plant tissues. Five A. arenosa root and shoot samples separately (twelve samples, three
standard solutions were prepared ranging from 0.05 to 10 ng μl−1. plants per sample were prepared) were dried at 105 °C with a moisture
analyzer RADWAG MAX 50/1 and quantitatively transferred to a vessel
2.6. RNA preparation with 5 ml of 65% solution of HNO3. The suspension was maintained for
1 h at ambient temperature and boiled for 1 h. After cooling, 1.650 ml
Total RNA was extracted from frozen in liquid nitrogen, ground of 30% hydrogen peroxide was slowly dropped into the solution and
leaves (from 3 plants per sample) with the Total RNA Mini Kit (Bio-Rad, heated until the start of the reaction (small bubbles in the suspension
US). RNA purity and quantity was determined by Biospec-Nano were observed). Subsequently, the suspension was centrifuged for
(SHIMADZU). The integrity of RNA was assessed with the Agilent 2100 15 min at 3000 rpm and the supernatant was quantitatively transferred
Bioanalyzer (USA) and RNA 6000 Nano Kit (Agilent, Germany). to a graduated 25 ml volumetric flask. The precipitate (if observed) was
treated with deionized water and vortexed. The resultant solution was
2.7. Microarray analysis centrifuged, and the supernatants pooled together. This procedure was
repeated twice. To verify the extraction efficiency the remaining pre-
To ensure optimal data quality, only RNA samples with RIN number cipitate was dried and tested for metal content with X-ray Fluorescence
≥8.5 were used in the analysis. Gene expression profiling was per- Spectroscopy.
formed using Arabidopsis (V4) Gene Expression Microarray, 4 × 44 K To determine metal concentration the calibration curve method was
(Agilent Technologies, USA) and Agilent Technologies Reagent Set ac- used. Zn and Fe were analyzed using FAAS, Pb and Cd were analyzed
cording to the manufacturer’s instruction. RNA Spike In Kit (Agilent using GF-AAS. P concentration was measured according to Margesin
Technologies, USA) was used as an internal control, the Low Input and Schinner (Margesin and Schinner, 2005). All standards were pur-
Quick Amp Labeling Kit −One Color was applied to amplify and label chased from Sigma Aldrich (Steinheim, D).

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2.11. Root hair development details can be found in Domka et al. (unpublished). Briefly, the fungus
colonizes both plant shoots and roots. The fungi enter root tissues by
For root hair growth evaluation Arabidopsis thaliana Col-0 (N6000), penetrating the root hairs, in their basipetal part and root epidermis.
etr1-3 (N237), eir1-1 (N8058), ein2-1 (N3071), aux1-7 (N9583), axr5-1 Aniline blue staining indicated intracellular localization of the fungi,
(N16234), tir1 (N3798), rhd6 (N6347) were used. All A. thaliana mu- however single hyphae were stained extracellularly, parallel to vascular
tants and transgenic lines were in the Col-0 background and were ob- tissues. On several occasions, fungal yeast like cells and lipid inclusions
tained from NASC (Nottingham Arabidopsis Stock Centre, UK). stained with Sudan IV were visualized inside vascular tissues and root
Seedlings were cultivated in in-vitro cultures as described previously. To hairs (Fig. 2A–H).
test the response of root hairs to the presence of toxic metals in the
substratum A. arenosa was grown in MSR supplemented with Pb, Zn and 3.2. Photosynthesis, water management is improved in E+ Arabidopsis
Cd (S3). Five days after inoculation roots were stained with 0.05% to-
luidine blue (O’Brien et al., 1964). Photographs of the primary root In order to verify if the Arabidopsis growth response is correlated
were taken with the stereomicroscope (Nikon SMZ 1500, JPN). For root with improved C assimilation we measured the rate of net photo-
hair number and length assessment a 5-mm fragment, 10 mm above the synthesis in E+ plants and compared it to non-inoculated plants.
root tip of the primary root was used. Measurements were performed on Inoculation with Mucor sp. resulted in a significant improvement in the
30 seedlings from 3 to 5 petri dishes per treatment/genotype. rate of CO2 assimilation. Net photosynthesis (PN) was increased by 33%
(Fig. 3A) Stomatal conductance (Gs) and the rate of transpiration of E+
2.12. Statistical analysis A. arenosa was improved by 43% and 53% respectively (Fig. 3B and C).
Additionally, water management was improved in endophyte in-
Data normality and variance homogeneity were evaluated by the oculated plants. The fresh to dry weight ratio of E+ A. arenosa was
Shapiro-Wilk and Levene’s tests, respectively. If not indicated other- significantly improved (Fig. 3D) indicating improved water uptake.
wise, statistical significance was determined with Student’s t-test
(p ≤ 0,05). Statistical analyses were conducted using STATISTICA ver. 3.3. Inoculation with Mucor sp. alleviates stress symptoms in A. arenosa
12 (Statsoft). A detailed description of statistical analysis in S5.
To assess the role of Mucor sp. in plant stress protection, the ex-
3. Results pression of stress related markers was evaluated. The concentration of
JA and ABA in roots of the plant was increased upon toxic metal
3.1. Mucor sp. improves A. arenosa growth. Visualization of A. arenosa treatment. Inoculation with the fungi significantly decreased the con-
colonization by Mucor sp. centration of all phytohormones measured. The most significant dif-
ference was found in SA concentration, where an app. 300-fold de-
E+ A. arenosa grown (Fig. 1A) mine dump substrate (Fig. 1A–C) crease was reported. Inoculation resulted in a 9-fold and 8-fold decrease
yielded significantly more biomass then E- plants. Five independent in JA and ABA concentration respectively (Fig. 3E).
experiments with 40–50 plants per experiment (in pot cultures with
different modes of inoculation and in vitro-Supplementary data) were 3.4. Transcriptomics and gene expression analysis
conducted to verify the growth response of the A. arenosa. In all cases
the E+ plants yielded significantly more biomass. To evaluate global changes in gene expression, microarray analysis
A detailed description of the colonization process and technical with the (V4) Gene Expression Microarray, 4 × 44 K (Agilent

Fig. 1. Fresh and dry weight of A. arenosa inoculated with Mucor sp. (E+; black bars) and not inoculated (white bars) grown in substrate from the mine dump “Bolesław” mixed with sand
in a 2:1. Statistical significance was evaluated with the nested ANOVA and the Tuckey post hoc test at P ≤ 0.05. Letters above bars indicate statistically significant differences; N = 3
(132 E+ and 115 control plants) (A). Seedlings were inoculated by application of 3.2 × 105 fungi spores in suspension to the substrate. Pictures of plants cultivated in a growth chamber
for 35 days (B, C).

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Fig. 2. Leaves (A–D) and roots (E–H) of A. arenosa

colonized by Mucor sp. stained with aniline blue and
sudan IV. (A–C) Fungal hyphae penetrating leaf sto-
mata (f), chloroplasts (c), mycelium entering stomata
(m). (D) Germinating spore of Mucor sp. (E) Fungal
mycelium (m) inside vascular tissue (v). (F)
Mycelium (m) inside root hair (h) and root cells. (G)
Fungal mycelium (m) inside root hair (h), lipid dro-
plets (l) stained with sudan IV close and inside my-
celium within root hair. (H) Hyphae visible between
root cells. Bars = 10 μm. (For interpretation of the
references to color in this figure legend, the reader is
referred to the web version of this article.)

Fig. 3. Net photosynthesis (A), stomatal conductance (B), transpiration rate Statistical significance was evaluated with the t-test at P ≤ 0.05. Stars above bars indicate statistically
significant differences. Gas exchange was measured on 10 plants. Salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) concentration in roots of the described above plants (E).
Statistical significance was evaluated with one-way ANOVA and the Tuckey post hoc test at P ≤ 0.05. Letters above bars indicate statistically significant differences, N = 5.

Technologies, USA) of roots of Mucor sp. colonized A. arenosa roots was probe sets; 2861 genes were up-regulated and 2308 down-regulated.
performed. A. arenosa Exhibits 5–10% DNA sequence variation in pro- Inoculation with the fungi induced changes in the expression of 899
tein-coding sequences (Chen et al., 2008), thus application of a tech- probes from the above presented data set; 492 genes were up-regulated
nology dedicated to A. thaliana is suitable for evaluating global gene and 314 were down-regulated. The expression of 93 genes (63 and 30,
expression. In order to distinguish between the effect of fungi coloni- up and down-regulated respectively) was affected by both treatments
zation and toxic metal stress on the plants transcriptome, we compared (Fig. 4A and B). Genes differentially expressed in E+ plants vs E- from
global gene expression of E- plants cultivated in control substrate mine dump substrate were subject to GO analysis (gene ontology) to
(commercially available soil mixed with sand in a 2:1 v:v ratio) with E- identify enriched terms with a cut-off rate of 0.01. For detailed results
A. arenosa grown in substrate from the mine dump. Subsequently, the see S2. The majority of the genes upregulated belonged to ROS meta-
obtained data set was compared to the gene expression profile of Mucor bolism, response to biotic and abiotic stimuli, response to ion starvation
sp. inoculated plants grown in the toxic metal enriched environment and ET biosynthesis and signaling functional categories. Among down-
(mine dump substrate). Metal toxicity altered the expression of 5169 regulated transcripts GO analysis revealed a significant enrichment in

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Fig. 4. Venn diagrams of up-regulated (A) and down-regulated genes (B) in A. arenosa roots inoculated with Mucor sp. (E + ) grown in substrate from the mine dump “Bolesław” mixed
with sand in a 2:1, 25 days after inoculation (TM). Differential gene expression was evaluated by comparing gene expression profiles of plants grown in control substrate, toxic metal
enriched substrate (TM) and TM inoculated with Mucor sp. Red circles represent the comparison between TME+ vs TM, blue circles TM vs control. Heat maps of selected enriched
functional categories representing changes in global gene expression (C). Statistical significance was assessed with the Student’s t-test, at least 2-fold up or down regulation at p ≤ 0.05.
For functional categorization SEA (Singular Enrichment Analysis) to identify enriched GO (gene ontology) terms with a false discovery rate (FDR) < 0.05 in Genespring 14.8 (Agilent
Technologies, USA) was performed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the regulation of anthocyanin biosynthesis term. Ethylene signaling, 3.5. The A. arenosa–Mucor sp. symbiosis accumulated less toxic metals but
and biosynthesis associated genes exhibited the most profound changes more P in its tissue. Root to shoots metal translocation was more efficient
in expression. Sixteen genes from the ET biosynthesis GO term in-
cluding ACO1 (ACC OXIDASE1; At2G19590), ACS9 (1-AMINOCYCLO- To confirm the role of Mucor sp. in metal distribution and phos-
PROPANE-1-CARBOXYLATE SYNTHASE9; At3G49700) and ACS11 (1- phorus acquisition, Fe, Zn, Cd, Pb and P concentration was measured in
AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE11; At4G08040) E+ and E- plants. Inoculation with Mucor sp. resulted in a lower ac-
were upregulated by the fungus as well as the expression of 21 genes cumulation rate of Zn and Fe; E+ plants accumulated 27% and 32%
associated with ET signaling (Fig. 4C). Most of the genes were upre- less Zn and Fe respectively. The concentration of Zn and Fe in the roots
gulated (17), however the abundance of 4 transcripts was decreased in of E+ A. arenosa was lower and reached 64% and 59% respectively. Zn
E+ plants including NAS1 (At5G04950) a nicotinamine synthase and Fe deposition in the shoots was increased, exceeding the con-
shown important in Fe homeostasis. A. arenosa inoculated with Mucor centration found in control plants by 70% and 32% respectively
sp. also exhibited a significant up-regulation in the expression of genes (Fig. 5A and C). Even though the accumulation rate of Cd was not
related with ion homeostasis (GO term response to nutrient level) metal significantly affected by the fungi, E+ plants translocated Cd from their
and phosphorous homeostasis in particular (Fig. 4C). Out of the 33 roots to shoots more effectively. The concentration of Cd in E+ A.
transcripts with altered abundance, only 2, the ammonium transporter arenosa shoots was increased by 88% (Fig. 5B). Mucor sp. did not affect
AMT1;4 (At4G28700) and the cytochrome P450 CYP7082A the quantity of Pb accumulated in plant tissues (Fig. 5D). The con-
(At5G48000) were down-regulated. Metal homeostasis genes that ex- centration of P was significantly, over 2-fold higher in E+ A. arenosa
hibited the most significant upregulation MTPA2 (METAL TOLERANCE (Fig. 5E).
PROTEIN A2; At3G58810), ZIP7 (Zn TRANSPORTER7 PRECURSOR; In addition to the metal carriers indicated to be affected by the fungi
At2G04032), IREG2 (IRON REGULATED2; AT5G03570) and IRT2 by microarray analysis, the expression of metal transporter genes that
(IRON REGULATED TRANSPORTER2; At4G19680). Additionally, 16 exhibited a significant increase according to the t-test with a false dis-
transcripts of genes related with P homeostasis were upregulated covery rate of 0.05 (less rigorous filtering) from the microarray data
(Fig. 4C), involved mainly in regulating the plants response to P were selected for verification with qPCR. HMA3 (HEAVY METAL
availability such as HRS1 (HYPERSENSITIVITY TO LOW PI-ELICITED ASSOCIATED3) is a tonoplast bound, P1B-2 type ATPase which partici-
PRIMARY ROOT SHORTENING1; At1G13300), SPX1 (SPX DOMAIN- pates in vacuolar Zn, Pb and Cd storage (Morel et al., 2009). Its ex-
CONTAINING PROTEIN1; At5G20150) and MONO- pression was significantly upregulated upon exposition to toxic metals.
GALACTOSYLDIACYLGLYCEROL SYNTHASE2 (MGD2; At5G20410). Inoculation with Mucor sp. resulted in its further upregulation. PCR2 is
a Zn transporter responsible for Zn extrusion and root to shoot Zn
translocation (Song et al., 2010). Its expression was significantly up-
regulated in E+ plants compared to A. arenosa grown in substrate from
the mine dump. ZINC-INDUCED FACILITATOR1 (ZIF1), a tonoplast

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P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

Fig. 5. Fe (A), Cd (B), Zn (C), Pb (D) and P (E) concentration in roots, shoots and whole A. arenosa inoculated with Mucor sp. grown in substrate from the mine dump “Bolesław” mixed
with sand in a 2:1. Statistical significance was evaluated with the t-test at P ≤ 0.05. Stars above bars indicate statistically significant differences, N = 12. Relative expression of selected
metal homeostasis related genes (F), quantified by qPCR. Statistical significance was evaluated with one-way ANOVA and the Tuckey post hoc test at P ≤ 0.05. Letters above bars indicate
statistically significant differences, N = 5. Grey bars indicate plants grown in control substrate (commercially available substrate mixed with sand in a 2:1 v:v ratio), white bars plants
grown on the mine dump “Bolesław” substrate mixed with sand in a 2:1 and black bars represent plants from mine dump “Bolesław” substrate inoculated with Mucor sp.

bound member of the major facilitator superfamily, transports Zn into 3.7. Ethylene and auxin are necessary for developing the root hair
the vacuole (Haydon and Cobbett, 2007). MTP1 (METAL TRANSPORT phenotype
PROTEIN1) encodes a Zn vacuolar transporter from the cation diffusion
transporter family. The expression of MTP1 was not affected in control ET perception is mediated by 5 homologous receptors, which appear
TM plants, however inoculation with Mucor sp. significantly increased largely redundant in function, although ETR1 (ETHYLENE
its expression. CAX2 (CATION EXCHANGER2) from the CAX- type fa- RESPONSE1) seems to play a dominant role in ET perception. EIN2
mily of antiporters plays a similar role in Cd transport as ZIF1 and (ETHYLENE INSENSITIVE1) is a positive regulator of ET signaling, acts
MTP1 (Koren’kov et al., 2007). The expression of CAX2 was sig- downstream of CTR1 (CONSTITUTIVE TRIPLE RESPONSE1), activating
nificantly decreased upon toxic metal treatment, however, inoculation EIN3/EIL (ETHYLENE INSENSITIVE3/ETHYLENE INSENSITIVE LIKE)
restored the expression to control values (Fig. 5F). (Eshel and Kafkafi, 2013). A. thaliana ethylene signaling mutants etr1
and ein2 did not develop the root hair phenotype, suggesting that ET is
necessary for fungi dependent root hair elongation. Auxin activates
3.6. Inoculation with Mucor sp. induces root hair elongation but does not transcription by targeting for degradation Aux/IAA transcriptional re-
affect other traits of the root pressors what leads to liberating ARF’s (auxin response factors) from
their non-functional dimerized with Aux/IAA state. Targeting for de-
To determine the role of plant growth regulators in Mucor sp. de- gradation is mediated upon interaction of IAA with TIR1 (TRANSPORT
pendent root hair elongation A. thaliana mutants were grown in INHIBITOR RESPONSE1) and formation of the SCFTIR1 complex
medium not supplemented with toxic metals. According to our data (Kepinski and Leyser, 2005). AXR5 (AUXIN RESISTANT5) is a member
(not shown) A. thaliana is not as tolerant to high quantities of TM in the of the Aux/IAA family. It interacts with TIR1 in an auxin-dependent
substratum as A. arenosa. Cultivation of seedlings in medium supple- manner resulting in AXR5 degradation. The mutation in AXR5 stabilizes
mented with Pb, Fe and Cd in 1/2 of the concentration used for A. the protein and leads to constitutive repression of auxin signaling (Yang
arenosa was lethal to the seedlings or caused 100% inhibition of seed et al., 2004). The root hair phenotype of tir1 and axr5 mutants was not
germination (data not shown). Nevertheless, the E+ root hair pheno- affected by inoculation with Mucor sp. indicating that for fungi de-
type was independent of toxic metals deposited in the substratum (Figs. pendent root hair elongation activation signaling is necessary. The role
6 A, S3), thus growth in regular medium was relevant to assess the root of the auxin influx carrier AUX1 (AUXIN RESISTANT1) in root hair
hair response. The length of root hairs of E+ A. arenosa and A. thaliana elongation is controversial (Jones et al., 2009), however, it was sug-
was significantly increased (nearly 2.5-fold). In response to toxic me- gested to affect the process (Lee and Cho, 2013). PIN2 (PIN-FORMED2;
tals, plants increase root hair density and length. In this study, root hair eir1) is an auxin efflux carrier localized in the plasmalemma of root hair
length increased by 2-fold upon toxic metal treatment and further, by and non-root hair cells. Its loss should alter the supply of auxin from the
3.5-fold in E + TM A. arenosa (Fig. 6A). Primary and lateral root root tip to the root differentiation zone and suppress root hair growth
growth was not affected by the fungus. (Jones et al., 2009; Lee and Cho, 2013). Inoculation with Mucor sp.
induced elongation of root hairs of aux1 and pin2 in a similar fashion as
in the WT (over 2-fold increase). The rhd6 (root hair deficient 6) mutant

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P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

Fig. 6. The root hair elongation response of Arabidopsis arenosa and A. thaliana ethylene and auxin mutants to Mucor sp. Root hair length was measured on a 5-mm fragment, 10 mm above
the tip of the primary root in 30 seedlings per species/genotype 5 days after inoculation, grown in MSR depleted of P. Statistical significance was evaluated with the t-test at P ≤ 0.05,
N = 3-5. A. arenosa seedlings grown on MSR medium (A) and supplemented with Fe, Cd, Zn and Pb (B). A. thaliana WT and etr1-3, eir1-1, ein2-1, aux1-7, axr5-1, tir1, rhd6 mutants (C)
inoculated with Mucor sp. Bar graph representing the root hair elongation response of Arabidopsis to Mucor sp. White bars represent control plants, black bars plants inoculated with Mucor
sp. Stars above bars represent statistically significant differences.

is defective in root hair initiation, it’s root hair less phenotype can be mg·kg−1, however, in contrary to the A. thaliana-C. tofieldiae model the
reversed by endogenous application of auxin and the ET precursor ACC effect of the fungi was P availability independent; growth of plants
(1-aminocyclopropane 1-carboxilic acid) (Masucci and Schiefelbein, under optimal conditions was significantly accelerated, indicating that
1994). With this in mind, we hypothesized that the fungi by activating Mucor sp. dependent growth improvement was not restrained to lim-
ET biosynthesis should induce root hair growth in rhd6 plants. Sur- iting conditions (S6). Nevertheless, transcriptomic data indicate that a
prisingly, we reported no root hair response of rhd6 mutants to in- phosphate starvation response (PSR) was activated in E+ plants. Ad-
oculation with the fungi, what confirms that the fungi do not affect root ditionally, the abundance of P was also higher in these plants. The
hair number but interferes solely with the elongation process (Fig. 6B expression of genes encoding inorganic pyrophosphatase 1
and C). (At1G73010), acid phosphatase/vanadium-dependent haloperoxidase-
related protein (At1G67600), phosphoethanolamine/phosphocholine
4. Discussion phosphatase (At1G17710) and glycerophosphoryl diester phospho-
diesterase family protein (At5G41080) all engaged in P liberation from
The beneficial impact of fungal endophytes on plant growth has intracellular sources was significantly increased. The substrate in which
been described previously (Brotman et al., 2013; Contreras-Cornejo plants were cultivated was fairly rich in organic P, unavailable for root
et al., 2009; Lee et al., 2011; Rozpądek et al., 2015; Varma et al., 1999) uptake unless hydrolyzed. Acid phosphatases, purple APases (PAP) in
and several efforts have been made to utilize these microorganisms in particular, are the predominant secretory APases which liberate P from
improving plant performance (Johnson et al., 2013; Kauppinen et al., organic sources. Out of the PAPases identified in A. thaliana, PAP12
2016). However, to our knowledge, a detailed description of the me- (purple acid phosphatase12) and PAP26 are the most highly expressed
chanisms of this mutualistic interaction has not been presented and (Duff et al., 1994; Plaxton and Tran, 2011). The expression of PAP12
even less is known about the role of fungal endophytes in plant adap- (At2G27190) was significantly upregulated in E+ suggesting that im-
tation to metal toxicity (Li et al., 2011, 2012; Wężowicz et al., 2014, proved P uptake was achieved by more efficient solubilization and
Wężowicz et al., 2017). Recent reports indicate that endophytic fungi subsequent uptake of P deposited in the rhizosphere. Additionally,
facilitate P uptake. Tichoderma asperellum was reported to improve PHO1;H1 expression induction indicates more efficient root to shoot P
phosphate uptake and acid phosphatase activity of its host cacao transport (Stefanovic et al., 2007). In plant-AMF, as well as in the A.
seedlings (Stewart and Hill, 2014). A similar phenomenon was observed thaliana- C. tofieldiae model, fungi hyphae extends out of rhizosphere
in the Neotyphodium coenophialum-Festuca arundinacea symbiosis into the surrounding soil and transmits water and nutrients into the
(Rahman and Saiga, 2005). Colletotrichum tofieldiae improved A. plant (Gutjahr and Parniske, 2013; Hiruma et al., 2016). Our results
thaliana growth under P starvation by transferring P to the plant by a suggest that in addition to the described above routs of P acquisition,
dense hyphae extending from inside root cells into the surrounding soil endophytic fungi may also stimulate plants to produce secretory en-
(Hiruma et al., 2016), similarly to mycorrhizal fungi (Plaxton and Tran, zymes that facilitate P uptake.
2011). The authors have shown that C. tofieldiae induces the expression Apart from improving the effectiveness of P acquisition, the water
of A. thaliana phosphate uptake and root to shoot translocation genes status of E+ A. arenosa was significantly better than non-inoculated
(Hiruma et al., 2016). A. arenosa suffered from P deficiencies, the plants. Stomatal conductance and the transpiration rate of E+
concentration of available P in the substrate was fairly low reaching 12 Arabidopsis was higher, indicating that E+ plants acquired water from

38
P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

the substrate more efficiently. To maintain an appropriate water status, shootward and rootward auxin transport (Negi et al., 2010). Auxin is
plants adjust transpiration by controlling stomatal aperture. Abscisic thought to operate both upstream of ET, stimulating its biosynthesis as
acid (ABA) plays a pivotal role in this process. In response to water well as downstream of ET (Muday et al., 2012). The role of auxin and
deficits, a rise in ABA production and subsequent initiation of ABA the possible auxin-ET interplay in Mucor dependent root hair elongation
signaling lead to stomatal closure, limiting water losses (McCourt and was investigated with auxin signaling and transporter (influx/efflux)
Creelman, 2008; Wilkinson and Davies, 2010). The concentration of mutants and DR5:GFP reporter lines (S1). Surprisingly, AUX1 nor PIN2
ABA in E+ plants was significantly lower, what supports the idea of deficiency and the resulting supply of auxin for the root hair differ-
improved water relations in E+ plants. Insufficient water and nutrient entiation zone did not prevent Mucor sp. induced root hair growth.
supply, what is commonly encountered in metalliferous environments, Previously AUX1/PIN2 dependent auxin flow through cells adjacent to
causes metabolic disturbances resulting in stress what consequently root hair cells was shown to be necessary for root hair growth (Jones
hinders plant growth. Besides improved biomass production, the ex- et al., 2009). We have also shown that no increase in the GFP reporter
pression of genes related to anthocyanin biosynthesis (hallmark re- signal was present in roots of E+ plants (S1) what contradicts with
sponse under P deficiency) was down-regulated in E+ Arabidopsis and previous reports (Contreras-Cornejo et al., 2009). These results suggest
the expression of RNS1 (ribonuclease1; AT2G02990) an anthocyanin that root hair phenotype was independent of auxin supply, however,
synthesis inhibitor was up-regulated upon inoculation. Additionally, root hairs of auxin signaling mutants tir1 and axr5 were insensitive to
the concentration of stress related hormones SA and JA was decreased inoculation indicating that even though auxin transport to root hair
in E+ plants. Stress alleviation by symbiotic fungi was previously re- cells was not necessary for elongation, maintaining auxin signaling was.
ported on several occasions (Li et al., 2011; Meier et al., 2011; Stein It cannot be ruled out that fungi derived auxin substitutes for the lack of
et al., 2008; Talaat and Shawky, 2014). Fungal endophytes such as P. supply of auxin to aux1 and pin2 mutants. Indeed, Mucor sp. in culture,
indica, S. vermifera and Epichloë festucae were also shown to directly could synthesize IAA, but the hormone produced by the fungi did not
affect plant hormone homeostasis by activating plant SA, JA metabo- affect the overall auxin status (S1).
lism or by producing SA-degrading enzymes respectively. Salicylic acid Toxic elements that surpassed barriers at the plant-soil interface and
degradation by fungal salicylate hydroxylase seems, however, unlikely entered plant tissues, are compartmented (cell compartment, tissue,
(Lahrmann et al., 2015; Ambrose et al., 2015). Based on the results of organ) and detoxified. Zhu et al. (2015) reported that two species be-
this study, we speculate that the stress protective role of the Mucor sp. longing to Mucor genus: M. circinelloides and M. racemosus improved
derives rather from its ability to improve plant water and nutrient biomass production and the accumulation of Pb and Cd of Guizhou
uptake then from exerting a direct effect on stress protective mechan- oilseed rape. Similarly, self-fusant protoplasts of Mucor sp. improved
isms. Improved photosynthesis efficiency which was previously pro- rape Cd and Pb tolerance and metal root to shoot translocation (Deng
posed to play a role in mutualistic fungi induced growth acceleration et al., 2013). Here, we have shown that in addition to more effective
(dek et al., 2015, 2014;) may be an analogous case. metal avoidance (E+ A. arenosa accumulated less Fe and Zn), root to
According to previous reports endophytic fungal symbionts induce shoot translocation of Cd, Fe and Zn was upregulated in inoculated
changes in root architecture (Gutjahr and Paszkowski, 2013; Stewart plants. This indicates that the symbiosis affected metal management
and Hill 2014; Hiruma et al., 2016). Trichoderma harzianum induced allowing the plant to accumulate less toxic elements in the roots and
maize root hair growth (Harman et al., 2004) and the endophytic Pir- more in its shoots, leading to a more uniform distribution of metals
iformospora indica and orchid mycorrhizal Sebacina vermifera were able between different organs. The elevated expression of genes associated
to induce lateral root development and growth in Arabidopsis. In the with metal transport and distribution such as TcZNT1, TcHMA4 and
case of both, the sebacinoid endophytes and the model used in this TcMTP1 and its homologs in A. hallerri and A. thaliana was suggested to
study, inoculation resulted in increasing the root absorption area and be crucial factors in determining metal tolerance (Becher et al., 2004;
consequently improving nutrient and water uptake, necessary for fungi Milner and Kochian, 2008). HMA4 and most likely HMA2 are the main
induced growth promotion. A. thaliana mutants with disturbed root hair transporters responsible for loading metal ions from the symplast into
development acquire less inorganic P (Bates and Lynch, 2000). It can be the xylem (Hanikenne et al., 2008; Haydon and Cobbett, 2007). The
speculated, that the physiological relevance of the fungi determined expression of these genes was not affected by the fungi. Another metal
root patterning and growth is the same in both models (the absorption transporter indicated in Zn phloem loading is PCR2 (Song et al., 2010).
area increases), but differs depending on the fungal symbiont inhabiting Its expression was upregulated by close to 2-fold in E+ plants com-
Arabidopsis. Root hair growth is controlled by the coordinated action of pared to the toxic metal control, suggesting that the increased root to
plant growth promoting factors including auxin and ET (Lee and Cho, shoot translocation was carried out by PCR2. Additionally, PCR2 was
2013; Muday et al., 2012; Song et al., 2016). Inoculation with Mucor sp. suggested to function in metal extrusion (Song et al., 2010), thus it
lead to a significant upregulation of genes involved in ET biosynthesis, cannot be ruled out that increased expression of PCR2 in E+ A. arenosa
signaling and response, indicating an upregulation in ET biosynthesis. resulted in lowering the concentration of metals in plant roots. Metal
Similarly, Chen et al. (2009) showed that the nectrotrophic fungus compartmentation into the vacuole is performed by numerous tono-
Fusarium graminearum exploits the ET pathway to aid infection of plast-bound transporters which have been reported to play an im-
Arabidopsis and barely. ET was found to inhibit primary root elongation, portant role in metal homeostasis and tolerance. Heterologous expres-
lateral root initiation and growth and the gravitropic response, whereas sion of HMA3 in the ycf1 yeast strain was shown to rescue the Pb/Cd
root hair development, growth and root waving was promoted by ET sensitive phenotype. Overexpression of this protein was suggested to
(Eshel and Kafkafi, 2013). ET signaling mutants etr1 and ein2 did not determine Cd, Pb and Zn tolerance in A. thaliana (Chao et al., 2012;
develop the root hair phenotype upon inoculation. This provides further Morel et al., 2009). MTP1 and MTPA2 play a similar role in Fe and Zn
evidence for the role of ET in determining the fungi induced root hair transport (Arrivault et al., 2006; Kawachi et al., 2009). Overexpression
response. of AtCAX2 in tobacco resulted in increased tolerance to Mn2+ and ac-
ET and auxin act synergistically in root hair elongation. Thirty eight cumulation of Cd2+ (Hirschi et al., 2000). ZIF1 from the major facil-
percent auxin responsive genes and 28% ET responsive genes were itator superfamily transporter (MFS) confers Zn tolerance in A. thaliana
shown to be regulated by ET and auxin respectively (Stepanova et al., probably by Zn transfer from the cytosol to the vacuole (Haydon and
2007). Auxin is also able to rescue root hair phenotypes in ET-in- Cobbett, 2007).
sensitive mutants (Rahman et al., 2002), suggesting that the action of
both of these phytohormones is necessary for root hair growth. Studies 5. Conclusion
with the exogenously applied ET precursor ACC and the ET over-
expressors eto1 and epi indicate that ET is a positive regulator of both The results presented in this study clearly indicate that the inherent

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P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

Fig. 7. Inoculation with Mucor sp. (E+) accelerates A. arenosa growth

in substrate polluted with high quantities of Fe, Zn, Pb and Cd. E+
plants assimilated CO2 and acquired water and phosphorus more ef-
ficiently. Water and nutrient uptake was facilitated by increased root
hair length and consequently the volume of soil penetrated by the
root. Up-regulated expression of genes coding proteins (PAP12,
At2G27190) by the fungi indicates that improved P acquisition relied
on liberating inorganic phosphate from insoluble resources and sub-
sequent uptake the plant.

Fig. 8. Arabidopsis arenosa inoculated with Mucor sp. grown in sub-

strate from mine dump “Bolesław” accumulates less Fe and Zn and
translocates Fe, Cd and Zn from its roots to the shoot more efficiently
than E- plants. This allows a more uniform distribution of toxic metals
within the plant. In E+ Arabidopsis the expression of several genes
engaged in metal transport into the vacuole was up-regulated. This
may explain improved toxic metal detoxification and tolerance of E+
plants.

mycobiota of A. arenosa attenuates the effects of metal toxicity, what Inoculation with Mucor sp. resulted in lower metal accumulation
implies a role in plant adaptation and succession in the extremely and significant alterations in metal distribution within the plant. This
hostile environment of a post mining waste dump. Inoculated plants was accompanied by upregulation of several metal homeostasis related
grown in substrate from the mine dump acquired P and water more genes. A large number of them facilitate metal transport from the cy-
efficiently probably due to fungi dependent root hair elongation, what tosol into the vacuole and determine toxic metal tolerance (Fig. 8).
significantly increased the volume of soil penetrated by the root.
Experiments with A. thaliana auxin and ethylene mutants and global Author contributions
gene expression profiling suggest that root hair elongation was driven
by a ET dependent mechanism and auxin, both plant and fungi derived, PR wrote the manuscript, planned and supervised the experiments;
played only a marginal role in the process. The expression of several AD planned experiments, performed experiments, editorial work; RW
genes related to PSR facilitated P uptake, however, P acquisition performed experiments, editorial work, MN performed experiments,
probably relied on a mechanism that differs from the one described prepared figures; RJ performed experiments, KT performed experi-
previously for mycorrhiza and other fungal endophytes (Fig. 7). ments, KT idea, performed experiments, editorial work

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P. Rozpądek et al. Environmental and Experimental Botany 147 (2018) 31–42

Acknowledgments Grierson, C., Schiefelbein, J., 2002. Root hairs. Arab. B 1, e0060. http://dx.doi.org/10.
1199/tab.0060.
Gutjahr, C., Parniske, M., 2013. Cell and developmental biology of arbuscular mycorrhiza
This work was supported by NSC, Maestro Project, DEC –2011/02/ symbiosis. Annu. Rev. Cell Dev. Biol. 29, 593–617. http://dx.doi.org/10.1146/
A/NZ9/00137. The authors would like to acknowledge Teresa Anielska, annurev-cellbio-101512-122413.
Martyna Janicka, Weronika Janas, Maciej Choczyński and Katarzyna Gutjahr, C., Paszkowski, U., 2013. Multiple control levels of root system remodeling in
arbuscular mycorrhizal symbiosis. Front. Plant Sci. 4, 204. http://dx.doi.org/10.
Wężowicz, PhD (Jagiellonian University, Poland) for technical support. 3389/fpls.2013.00204.
Hanikenne, M., Talke, I.N., Haydon, M.J., Lanz, C., Nolte, A., Motte, P., Kroymann, J.,
Appendix A. Supplementary data Weigel, D., Krämer, U., 2008. Evolution of metal hyperaccumulation required cis-
regulatory changes and triplication of HMA4. Nature 453, 391–395. http://dx.doi.
org/10.1038/nature06877.
Supplementary data associated with this article can be found, in the Harman, G.E., Petzoldt, R., Comis, A., Chen, J., 2004. Interactions between Trichoderma
online version, at https://doi.org/10.1016/j.envexpbot.2017.11.009. harzianum strain T22 and maize inbred line mo17 and effects of these interactions on
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