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Metabolic changes induced by manganese in chamomile
Accepted Manuscript Metabolic changes induced by manganese in chamomile Jozef Kováčik, Sławomir Dresler, Magdalena Wójciak-Kosior, Juraj Hladký, Petr Babula PII:
S0981-9428(18)30477-7
DOI:
https://doi.org/10.1016/j.plaphy.2018.10.031
Reference:
PLAPHY 5473
To appear in:
Plant Physiology and Biochemistry
Received Date: 2 September 2018 Revised Date:
28 October 2018
Accepted Date: 28 October 2018
Please cite this article as: J. Kováčik, Sł. Dresler, M. Wójciak-Kosior, J. Hladký, P. Babula, Metabolic changes induced by manganese in chamomile, Plant Physiology et Biochemistry (2018), doi: https:// doi.org/10.1016/j.plaphy.2018.10.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Metabolic changes induced by manganese in chamomile
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Jozef Kováčik
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Babula e
a*
, Sławomir Dresler b, Magdalena Wójciak-Kosior c, Juraj Hladký d, Petr
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a
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Republic
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b
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033 Lublin, Poland
Department of Biology, University of Trnava, Priemyselná 4, 918 43 Trnava, Slovak
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Department of Plant Physiology, Maria Curie-Skłodowska University, Akademicka 19, 20-
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c
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Lublin, Poland
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d
Faculty of Education, University of Trnava, Priemyselná 4, 918 43 Trnava, Slovak Republic
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e
Department of Physiology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625
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00 Brno, Czech Republic
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corresponding author e-mail: [email protected]
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Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093
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ACCEPTED MANUSCRIPT ABSTRACT
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Manganese (Mn) uptake and toxicity in chamomile (Matricaria chamomilla) and changes of
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phenolic metabolites in plants grown in the soil (1000 µM Mn2+) or hydroponic culture (100
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or 1000 µM Mn2+) were studied. Under soil cultivation, Mn excess reduced growth and
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induced symptoms of oxidative stress (including total ROS, hydroxyl radical and lipid
23
peroxidation as detected by fluorescence microscopy), concomitantly with depletion of non-
24
protein thiols and ascorbic acid. Total soluble phenols and individual phenolic acids were
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rather depleted (p-coumaric, chlorogenic, and protocatechuic acids) or unaltered (vanillic and
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caffeic acids). Shoot Mn content reached 2806 µg/g DW with BAF 51.0 in the soil culture. In
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hydroponics, tetraploid plants contained less Mn in both shoots and roots than diploid ones
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with bioaccumulation factor and translocation factor (diploid/tetraploid) 57.1/37.9 and
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0.39/0.32 in 1000 µM Mn treatment. Plants cultured in hydroponics revealed stimulation of
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some phenolic acids, mainly chlorogenic acid in the shoots and p-hydroxybenzoic and vanillic
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acids in the roots (more extensively in tetraploid ones which contained less Mn). Data
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indicate that excessive Mn accumulation has negative impact not only on the growth but also
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on phenolic metabolites in young plants mainly. Detailed comparison of the observed
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metabolic changes with limited literature focused on Mn physiology is provided as well.
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Keywords: antioxidants; fluorescence microscopy; heavy metals; soil pollution.
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ACCEPTED MANUSCRIPT 1. Introduction
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Manganese (Mn) is an essential plant micronutrient which is a component of e.g. numerous
40
enzymes (Millaleo et al., 2010). It naturally occurs in the soil but its elevated levels are
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detectable in polluted soils (Kandziora-Ciupa et al., 2013). Its uptake is relatively fast process
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in various crop or weed species (Kováčik et al., 2014b; Inostroza-Blancheteau et al., 2017)
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and is typically higher compared to other metals including also data from chamomile
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(Matricaria chamomilla; Kováčik et al., 2010 and 2014a). As in the case of other metals, Mn
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toxicity is variable even in genotypes of the given species with subsequent variability in the
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growth and metabolic responses (Dziwornu et al., 2018). Clear excluders or Mn accumulators
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are therefore known with translocation factor over or below 1 (Kováčik et al., 2014a;
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Inostroza-Blancheteau et al., 2017).
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Toxicity of metal excess is typically evoked by elevated reactive oxygen species
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(ROS) formation which is relatively mild under Mn excess if assayed by standard
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spectrophotometry (i.e. low increase in hydrogen peroxide or MDA content) as observed in
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several species (Farzadfar et al., 2017; Nazari et al., 2017). On the contrary, fluorescence
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microscopy as a more sensitive technique for oxidative stress detection (Kováčik and Babula,
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2017) revealed considerable enhancement of ROS and lipid peroxidation in various plants
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(Kováčik et al., 2014a and 2014b). This metal-induced oxidative stress must be regulated by
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various mechanisms to prevent damage and eventual lethal impact on the growth. They
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include both antioxidative enzymes and non-enzymatic antioxidants. Enzymatic activities are
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typically elevated (Gangwar et al., 2010; Santos et al., 2017) while non-enzymatic
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antioxidants such as ascorbic acid (AsA) and non-protein thiols including glutathione are
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rather depleted by Mn excess in plants (Shi et al., 2006; Gangwar et al., 2010). Phenolic
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metabolites as widespread secondary plant products were relatively rarely been monitored
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under Mn excess. Total soluble phenols show rather slight elevation under moderate Mn
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excess (Farzadfar et al., 2017) and more pronounced elevation under higher Mn doses
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(Inostroza-Blancheteau et al., 2017). At the level of individual metabolites, phenolic acids
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revealed rather negligible changes in the shoot tissue (Farzadfar et al., 2017) or in fruits
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(Vithana et al., 2018). Chamomile is a widely used medicinal plant showing often tolerance to higher metal
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concentrations and genotype-related pattern of the accumulation of some metals (Kováčik et
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al., 2010 and 2012). Previous work revealed higher sensitivity of chamomile seedlings (in
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comparison with older plants cultured in hydroponics) to Mn excess at the level of growth,
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while symptoms of oxidative stress were rather similar (Kováčik et al., 2014a). No metabolic
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responses were monitored in the previous studies and we therefore compared Mn excess in
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the soil and in hydroponics in terms of Mn accumulation and the Mn impact on phenolic acids
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in chamomile. In addition, ROS appearance and comparison of genotypes were monitored.
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Data are explanatively compared with related studies in terms of similarities and differences
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of Mn-induced metabolic changes.
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2. Materials and methods
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2.1. Cultivation, experimental design and statistics
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Soil experiment was performed in Eutric Cambisol (with sandy loamy texture) containing
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water-soluble Mn ca. 1.50 mg/kg (Kováčik et al., 2014b) marked as control soil in the present
82
study. Parallel identical soil was enriched with Mn (as MnCl2.4H2O) to achieve 1000 µM Mn
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(55 mg Mn/kg), marked as Mn soil in results. This concentration is ca. 1/5 from water-soluble
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Mn content in naturally-contaminated Mn soil which evoked strong growth depression of
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several crops in the previous work (Kováčik et al., 2014b). Soil moisture was maintained at
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60% of water holding capacity with distilled water and no additional nutrients were applied
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(pot diameter 10 cm with 0.5 kg of the soil). Cultivation was realized under laboratory
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tubes L36W/840 (Lumilux, Osram), 25/20°C day/night temperature and relative humidity of
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~60 % (Kováčik et al., 2014a). Tetraploid chamomile (Matricaria chamomilla cv. Lutea,
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Asteraceae) seeds were sown directly on the surface of the soil (20 seeds per pot, 20 pots for
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each treatment) and germinated within 48 – 72 h. Seedlings were cultured for additional 20
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days when the growth responses between control and Mn-enriched soil became visible.
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Owing to tiny roots, only above-ground biomass was analyzed for minerals and biochemical
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parameters. Seedlings from one pot were pooled to achieve enough biomass for individual
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measurements. For fresh mass-requiring parameters, samples were extracted with cold mortar
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and pestle as mentioned below and dry samples (dried at 75°C to constant weight) were
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analyzed for mineral nutrients and phenols.
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In the subsequent experiment, diploid and tetraploid chamomile plants were pre-
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cultured in the sand followed by 4 weeks of cultivation in hydroponics with Hoagland
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solution (Kováčik et al., 2014a). Thereafter, plants were exposed to 100 or 1000 µM Mn (as
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MnCl2.4H2O), control contained 2.03 µM Mn2+ as micronutrient and pH was checked to be
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6.0 in all treatments. One 2-L box containing 10 plants was used for each treatment with two
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repetitions, thus the whole experiment included 12 boxes. After 10 days of cultivation with
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Mn excess, shoots and roots were separated, roots washed trice with deionised water and
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dried (at 75°C to constant weight) for the assay of Mn accumulation and phenolic metabolites.
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Student’s t-test was used to compare the differences between control and Mn-enriched
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soil. Data from hydroponics were evaluated using ANOVA followed by a Tukey’s test
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(MINITAB Release 11, Minitab Inc.; State College, Pennsylvania) at P<0.05 for diploid or
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tetraploid samples individually while Student’s t-test was used to determine difference
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between diploid and tetraploid samples in the given treatment.
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Samples were prepared by mineralization of dry plant material (50 mg) in the mixture of
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concentrated HNO3 and water (5 + 5 mL) using microwave decomposition (Ethos Sel
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Microwave Extraction Labstation, Milestone Inc.) at 200°C over 1 h (complete duration of the
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mineralization program). Resulting clear solution was placed to inert plastic flasks and diluted
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to a final volume of 20 mL. All measurements were carried out using an atomic absorption
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spectrometer (Polarised Zeeman Z – 8200, Hitachi, Tokyo, Japan) as reported previously
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(Kováčik et al., 2014a; Dresler et al., 2017).
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2.3. Assay of enzymatic and non-enzymatic antioxidants
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For the assay of enzymatic activities, whole shoots were homogenized in potassium
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phosphate buffer containing 1% insoluble PVPP (pH 7.0) using cold mortar and pestle with
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the addition of small amount of inert so-called sea sand (Penta s. r. o., Prague, Czech
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Republic) to achieve complete tissue disruption, followed by centrifugation at 14 000 g for 15
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min at 4°C. Soluble proteins were quantified according to Bradford method and bovine serum
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albumin as standard (595 nm) as reported previously (Kováčik et al., 2014b). Ascorbate
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peroxidase (APX) and guaiacol peroxidase (GPX) activities were measured as the oxidation
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of ascorbate and guaiacol at 290 and 470 nm, respectively; glutathione reductase (GR)
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activity was assayed as the reduction of GSSG at 412 nm and catalase (CAT) activity as the
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reduction of H2O2 to water at 240 nm (Kováčik et al., 2014a and 2014b). Randomly selected
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supernatants were boiled and assayed in order to check that reactions were enzymatic.
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For the assay of ascorbic acid (AsA) and non-protein thiols (NPT), samples were
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extracted in 0.1 M HCl (0.1 g FW/mL) using cold mortar and pestle followed by
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centrifugation
as
mentioned
above.
Reduced
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AsA
content
was
determined
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bathophenanthroline method and NPT by 5,5ʼ-dithiobis-(2-nitrobenzoic acid) as reported in
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detail previously (Kováčik et al., 2017).
139 2.4. Fluorescence microscopy
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For microscopic analyses, cotyledons (the oldest photosynthetic part of the shoot) were used.
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Whole individual fresh cotyledons were immediately stained and observed. General
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accumulation of ROS was monitored using widely-used CellROX® Deep Red Reagent
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(644Ex/665Em
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radical/peroxynitrite reagent (aminophenyl fluorescein, 490Ex/515Em nm, Sigma-Aldrich).
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Nitric oxide or reactive nitrogen species (RNS) were visualized with 2,3-diaminonaphthalene
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(DAN, 365Ex/415Em nm, Sigma-Aldrich) and lipid peroxidation by BODIPY® 581/591 C11
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lipid peroxidation sensor (581Ex/591Em nm, Life Technologies, USA). Staining procedures
149
were conducted as reported previously (Kováčik et al., 2014a; Kováčik and Babula, 2017).
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Staining solution was always removed by respective buffer and samples were observed using
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fluorescence microscope (Axioscop 40, Zeiss, Germany) and appropriate set of filters.
Life
Technologies,
USA)
and
specifically
with
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2.5. Assay of phenolic metabolites
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Total soluble phenols were extracted with 80% methanol (100 mg DW/mL) and quantified
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using Folin-Ciocalteu method with gallic acid as standard and detection at 750 nm (Kováčik
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et al., 2014b).
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For the assay of individual phenolic acids, methanol extracts mentioned above were
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extracted twice with anhydrous diethyl ether, evaporated to dryness using SpeedVac and
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dissolved in 0.1 mL of 80% aqueous methanol. Root cell wall-bound phenols were measured
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after alkaline hydrolysis of methanol-insoluble root residue: samples were washed trice with
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methanol until no soluble phenols were detectable. Then they were treated with 1 M NaOH
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with diethyl ether, evaporated to dryness and dry residue was dissolved in 80% methanol as
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mentioned above (Kováčik et al., 2011). Quantification was done by HPLC/CE system and
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identification by comparison of the retention time and absorption spectrum similarity between
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the standards (all from Sigma-Aldrich) and the samples for the detectable phenolic acids as
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reported previously (Dresler et al., 2017; Sowa et al., 2018).
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3. Results and discussion
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3.1. Impact of soil Mn on its accumulation and mineral nutrients
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Water-soluble Mn content in control soil (ca. 1.50 mg/kg = µg/g) is similar to earlier study
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from Poland (1.70 mg Mn/kg; Nadgórska-Socha et al., 2013) but lower compared to other
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Polish sites (average value 4.0 – 9.7 mg Mn/kg; Kandziora-Ciupa et al., 2013). Under these
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conditions, control chamomile shoots contained 62.4 µg Mn/g DW which is similar to
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previous work where several crops contained 53 – 66 µg Mn/g DW in control soil (Kováčik et
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al., 2014b). Addition of Mn to soil (55 mg/kg) led to shoot Mn content over 2800 µg/g DW
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(Fig. 1). This bioavailable Mn content is higher compared to “naturally” polluted localities in
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Poland where 3.54 – 24.89 mg Mn/kg soil was reported (Kandziora-Ciupa et al., 2013): under
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these conditions, Vaccinium leaves contained 38.8 – 398 µg Mn/g DW, which is far lower
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compared to our present data. On the contrary, water-soluble soil Mn content 280 mg/kg in an
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earlier study led to shoot Mn amount 2300 – 13 600 µg/g DW in various crops (Kováčik et
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al., 2014b). In rice, higher Mn availability in the soil led to higher shoot Mn content (up to ca.
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4000 µg/g DW; Dziwornu et al., 2018).
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Owing to variability in soil and tissue Mn content, bioaccumulation factor is more
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suitable for comparison between studies. Shoot bioaccumulation factor (BAF) is defined as
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the ratio of shoot Mn content (µg/g DW) to soil Mn content (µg/g DW), i.e. 1.50 (control soil)
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for chamomile and are typically higher than those observed in various crops exposed to Mn
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excess: 8.4 – 48.9 (using shoot Mn content/water-soluble soil Mn content; Kováčik et al.,
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2014b). Few other studies also reported Mn uptake in plants from natural localities and shoot
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Mn BAF can be calculated. For example, shoot BAF in Vaccinium myrtillus (considering
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potentially bioavailable soil Mn fraction extracted with 0.01 M CaCl2) was lower in the soil
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from polluted localities (average value 17 for various time of harvest) in comparison with
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control localities (average value 140 for various time of harvest; Kandziora-Ciupa et al.,
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2013). On the contrary, Plantago lanceolata and Cardaminopsis arenosa from polluted
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locality (mining and metallurgic activity, bioavailable Mn was only up 3-times higher in
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polluted soil), revealed similar shoot BAF if compared to control site (average value 4.35 in
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P. lanceolata polluted sites vs. 3.6 in control; average value 11.8 in C. arenosa polluted sites
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vs. 14 in control; Nadgórska-Socha et al., 2013). All these data confirm that various plant
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species have various trends of Mn accumulation and that chamomile (considering typically
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higher shoot BAF both in control and Mn soil) has higher Mn accumulation potential in
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comparison with mentioned species.
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Excess of Mn, as in the case of excess of other essential or non-essential metallic ions,
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affects sorption of various nutrients. It was surprising to find that K and Zn content remained
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unaltered by excessive Mn accumulation (Fig. 1) as previously observed in hydroponically
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cultured chamomile (Kováčik et al., 2014a) and partially in some crops cultured in naturally-
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contaminated Mn soil (Kováčik et al., 2014b). In agreement, soybean exposed to 100 – 300
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µM Mn showed slight or no change in K, Ca, Mg, Zn and Cu content; interestingly,
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movement of Ca from the healthy area to necrotic area (evoked by Mn excess) and the
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opposite pattern of K movement were reported (Santos et al., 2017). On the contrary,
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chamomile in the present study showed depletion of Ca and Mg amount (Fig. 1) as previously
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indicating that responses are evoked rather by applied Mn concentration than by the mode of
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cultivation. Strong depletion of Ca and Mg content has also been found in crops cultured in
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naturally-contaminated Mn soil (Kováčik et al., 2014b) and it seems that Mn excess is a
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strong competitor of divalent cations (mainly Ca and Mg). In agreement with this assumption,
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Mn-induced (increase from 9.1 µM in control to 150 µM Mn) depletion of Mg in Tanacetum
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parthenium was reversed by an increase in exogenous Mg dose from 1 mM (= 1000 µM) in
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control to 2 – 4 mM and Mn accumulation decreased with increasing Mg dose (Farzadfar et
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al., 2017). Amount of cadmium, an example of non-essential and toxic metal, was lower in
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control chamomile (Fig. 1) than in the previously tested crops (Kováčik et al., 2014b) and Mn
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excess stimulated Cd accumulation.
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3.2. Impact of soil Mn on oxidative stress and antioxidants
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Mn-induced depression of the growth and soluble proteins of chamomile (Fig. 2) was also
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previously observed in the crops cultured in the soil with 280 mg water-soluble Mn/kg
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(Kováčik et al., 2014b). Other species are yet more sensitive to Mn excess such as Pisum
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sativum cultured with 100 – 250 µM Mn (Gangwar et al., 2010) or soybean cultured with 10 –
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300 µM Mn (Santos et al., 2017) while grass Lolium perenne (accumulating Mn considerably)
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was resistant to even 750 µM Mn (Inostroza-Blancheteau et al., 2017).
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Sensitivity of the given species reflected in the growth depression could be evoked,
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among other, by changes in the ROS balance (leading to damage of essential biomolecules if
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excessive ROS formation is not effectively controlled). In agreement, depletion of APX and
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GR activities may be related to depleted accumulation of ascorbic acid and non-protein thiols
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in response to Mn excess (Fig. 2). On the contrary, GPX and CAT activities were stimulated
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by Mn excess, as previously observed in crops cultured in naturally-contaminated Mn soil
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ACCEPTED MANUSCRIPT (Kováčik et al., 2014b). In agreement with our observations, Mn excess (600 µM) variously
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affected CAT, APX, GPX and GR in cucumber (Shi et al., 2006) or in pea cultured with 100 –
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250 µM Mn (Gangwar et al., 2010). On the contrary, all antioxidative enzymes showed
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elevation in response to 10 – 300 µM Mn in soybean but it is questionable whether the unit of
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activity at the level of mmol/min/mg protein is realistic (Santos et al., 2017). These data
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indicate that antioxidative enzymes are variously affected by Mn excess, leading to alteration
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of ROS balance. Fluorescence microscopy confirmed considerable elevation of both general
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ROS and hydroxyl radical in response to Mn, concomitantly with elevated lipid peroxidation
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and nitric oxide signal (Fig. 3). All these responses are similar to those observed in
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chamomile cultured in hydroponics with 100 – 1000 µM Mn (Kováčik et al., 2014a) and
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confirm responses of oxidative symptoms to Mn excess independently on the ontogenetic
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stage. In agreement, crops cultured in the soil with water-soluble Mn 280 mg/kg (ca. 5 mM)
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showed considerably enhanced ROS, NO and lipid peroxidation signal which may be a reason
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for suppressed growth (Kováčik et al., 2014b). On the contrary to our qualitative data
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showing strong enhancement of oxidative stress symptoms under Mn excess, quantitative data
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revealed significant but rather mild increase in H2O2 and superoxide accumulation in
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Tanacetum parthenium exposed to 150 µM Mn (Farzadfar et al., 2017) or in Mentha aquatica
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treated by 160 µM Mn (Nazari et al., 2017). In addition, only slight increase in lipid
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peroxidation has been observed in rice (we note that unit of MDA content nmol/mL/g is
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unclear; Dziwornu et al., 2018) and mainly Mn dose 750 µM elevated strongly MDA level in
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Lolium
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spectrophotometry is common in the literature, it does not reach sensitivity of the
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fluorescence microscopy to detect slight changes in the ROS formation (see Kováčik and
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Babula, 2017 for details).
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perenne
(Inostroza-Blancheteau
et
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2017).
Overall,
though
standard
ACCEPTED MANUSCRIPT Depletion of ascorbic acid and non-protein thiols in response to Mn excess could be a
262
reason for enhancement of oxidative stress appearance (cf. Figs 2 and 3) and growth
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inhibition as mentioned above and the same was previously observed in the cucumber
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exposed to 600 µM Mn (Shi et al., 2006) or in the pea plants treated by 100 – 250 µM Mn
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(Gangwar et al., 2010). Data from field-collected plants growing on the soil with various
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metallic contaminations including higher Mn amount showed variously affected glutathione
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and non-protein thiol contents: Mn revealed negative correlation with GSH in Vaccinium
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myrtillus (Kandziora-Ciupa et al., 2013) or in Cardaminopsis arenosa (Nadgórska-Socha et
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al., 2013) while non-protein thiols did not. We note, however, that GSH content in the two
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mentioned species cannot reach up to 250 µmol/g FW (= over 760 mg/g DW considering
271
tissue water content 90%) and is in strong contradiction to non-protein thiol content up to 1.8
272
µmol/g FW (as GSH is one of them). It is concluded that management of thiols and ascorbate
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accumulation in plants, to prevent their depletion, could provide a tool for better growth
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performance under Mn excess.
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3.3. Impact of solution Mn on its accumulation
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We note that applied Mn doses did not suppress growth of hydroponically cultured plants
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owing to longer pre-cultivation prior to Mn application while seedlings are more sensitive as
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reported previously (Kováčik et al., 2014a) and in this work (Fig. 2). Assay of Mn content in
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plants cultured in hydroponics revealed preferential Mn accumulation in the roots compared
281
to shoots except for control (Fig. 4). Ten fold increase in exogenous Mn (from 100 to 1000
282
µM) evoked ca. 5-6-fold and ca. 4-fold increase in shoot and root Mn accumulation,
283
respectively. These data indicate that chamomile readily absorbs Mn to shoots (over 400 µg
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Mn/g DW at 100 µM Mn) in comparison with e.g. soybean (ca. 200 µg Mn/g DW at 100 µM
285
Mn; Santos et al., 2017), Juncus effusus (137 µg Mn/g DW at 100 µM Mn; Najeeb et al.,
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contrary, related species Tanacetum parthenium (Asteraceae family too) contained more Mn
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in shoots (1898 µg Mn/g DW at 150 µM Mn with normal/1 mM Mg in the solution; Farzadfar
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et al., 2017) and grass species Lolium perenne revealed the same (842 – 938 µg Mn/g DW at
290
150 µM Mn; Inostroza-Blancheteau et al., 2017). It was observed that tetraploid chamomile
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contained less Mn in both shoots and roots than diploid ones under Mn excess (Fig. 4). In
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agreement, inflorescences of field-grown tetraploid chamomile contained less Cd, Hg, Cr, Cu,
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Al, Mg, Ca and K than diploid flowers (Kováčik et al., 2012) and lower Cd but not Ni
294
accumulation was observed in chamomile vegetative tissue (Kováčik et al., 2010). Ploidy
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level of chamomile may therefore affect accumulation of various nutrients which could be
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usable in selection of cultivars with lower metallic contamination (for pharmaceutical
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purposes) or with higher metal accumulation (for remediation purposes).
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As mentioned above, shoot BAF values are also calculable for solution culture as
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shoot Mn (µg/g DW)/solution Mn (µg/mL) ratio (from the Fig. 4, diploid/tetraploid plants):
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21.1/23.4 (control), 85.8/73.1 (100 µM Mn treatment) and 57.1/37.9 (1000 µM Mn
301
treatment). Value in tetraploid shoots in 1000 µM Mn treatment (37.9) is lower than in soil-
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cultured plants (51.0, see above) but is not considerably lower considering totally different
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mode of cultivation. It is wort noting that lower BAF values also reflect lower Mn content in
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tetraploid shoots (in comparison with diploid ones) and ploidy-dependent pattern is therefore
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visible as mentioned above. For comparison, shoot BAF value of Mn in soybean cultured with
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100 µM Mn is lower (36, calculable from the data by Santos et al., 2017) and reflects lower
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shoot Mn content too.
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On the contrary to BAF which reflects tissue metal content in relation to exogenously
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applied metal dose, translocation factor (TF) is a relative unit of shoot metal (µg/g DW) to
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root metal content (µg/g DW) ratio and indicates tendency of root to shoot metal movement.
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ACCEPTED MANUSCRIPT In chamomile, TF values (calculable from the Fig. 4) are (diploid/tetraploid plants) 2.1/2.6
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(control), 0.21/0.26 (100 µM Mn treatment) and 0.39/0.32 (1000 µM Mn treatment). These
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data are in agreement with previous report from tetraploid plants cultured in hydroponics (TF
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0.31 and 0.34 in 100 and 1000 µM Mn treatment) while seedling cultured with water Mn
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solution showed higher TF (0.57 and 0.47 in 100 and 1000 µM Mn treatment; Kováčik et al.,
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2014a). Surprisingly, species with lower shoot Mn content in comparison with chamomile
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may have higher TF such as Juncus effusus (TF 0.73 at 100 µM Mn; Najeeb et al., 2009) or
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soybean (TF>1 at 100 – 300 µM Mn; Santos et al., 2017), confirming excluder character of
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chamomile (i.e. preferential Mn accumulation in the roots). However, chamomile could be
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more suitable for eventual accumulation (in the shoots) and phytostabilization (root
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accumulation) of Mn ions.
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3.4. Impact of soil vs. solution Mn on phenols
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Phenolic metabolites are widespread secondary plant products involved also in responses to
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metallic stress as previously observed using specific inhibitor just in chamomile (Kováčik et
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al., 2011). Slight but significant depletion of total soluble phenols (common abbreviation
327
TPC) in the shoots of plants cultured in Mn soil (Fig. 2) indicates negative impact of Mn
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excess specifically in chamomile because higher soil Mn dose in the previous study did not
329
deplete soluble phenols in several crops (Kováčik et al., 2014b). In agreement, TPC content
330
was rather elevated by Mn excess 50 – 150 µM in Tanacetum parthenium (Farzadfar et al.,
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2017) or by higher Mn doses (350 or 750 µM) in Lolium perenne (Inostroza-Blancheteau et
332
al., 2017). In the fruits, exogenous application of 0.2 or 0.3% Mn2+ had negligible impact on
333
TPC in mango (Vithana et al., 2018). Subsequent assay of individual phenolic acids in
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chamomile shoots revealed their unaltered or depleted accumulation (as TPC also did), only
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p-hydroxybenzoic acid increased (Fig. 5). Is seems that Mn-induced oxidative stress in soil-
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cultured plants was strong enough to suppress not only general antioxidants (ascorbate and
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thiols) but also phenolic metabolites. In the hydroponically-cultured plants, chlorogenic acid was the only compound
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significantly enhanced by Mn excess both in diploid and tetraploid shoots while other acids
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remained unaffected (Table 1). In the roots (free fraction), p-hydroxybenzoic and vanillic
341
acids increased in both genotypes while chlorogenic acid in tetraploid roots only (Table 1).
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Various responses of chamomile genotypes at the level of phenolic metabolites have been
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previously reported (Kováčik et al., 2010). Chlorogenic acid is an important antioxidant
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which is often elevated by various stressors including metals such as Cd in chamomile
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(Kováčik et al., 2011) and its elevation could indicate the same function under Mn excess too.
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However, stimulation by Mn excess was lower in comparison with other metals. In
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agreement, only gallic and ferulic acids increased in response to 50 or 150 µM Mn in
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Tanacetum parthenium while other phenolic acids (p-hydroxybenzoic, salicylic, caffeic and
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chlorogenic acids) remained unaltered (Farzadfar et al., 2017): it is wort noting that many
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acids increased after application of elevated Mg dose which suppressed Mn uptake, thus
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probably preventing toxic impact of Mn on phenolic biosynthesis. This is indirectly
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confirmed by our present data because plants cultured in the soil revealed growth retardation
353
and depleted Mg content (Figs 1 and 2). Cell wall-bound acids were rather depleted under Mn
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excess and more extensively in diploid roots, indicating genotype-related differences (Table
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1). Significance of this phenomenon remains to be elucidated but it was previously observed
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also under Cd excess (Kováčik et al., 2011).
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Conclusions
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Present study showed that the growth retardation induced by soil Mn excess is related to the
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appearance of oxidative stress and depletion of antioxidants (not only ascorbic acid and thiols
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ACCEPTED MANUSCRIPT but also phenolic metabolites) with shoot Mn bioaccumulation factor 51.0. In hydroponics,
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tetraploid shoots in 1000 µM Mn treatment showed BAF 37.9 which is lower than in soil-
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cultured plants, but is not considerably low considering totally different mode of cultivation.
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Higher accumulation of Mn in both shoots and roots of diploid plants has also been recorded.
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Since no growth retardation was found, plants cultured in hydroponics revealed stimulation of
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some phenolic acids, mainly chlorogenic acid in the shoots and p-hydroxybenzoic and vanillic
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acids in the roots. It seems that excessive Mn accumulation has rather negative impact on
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phenolic metabolites in young plants mainly.
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369 Acknowledgement
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The work was supported by Slovak grant agency VEGA (project no. 1/0041/18).
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372 Disclosure statement
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The authors declare that there are no conflicts of interest.
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Role of the funding source
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Sponsor had no involvement in the present study.
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378 Contribution
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Experimental design, spectrophotometry and manuscript preparation (JK and JH), analyses of
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minerals and phenolic acids (SD and MWK), fluorescence microscopy (PB).
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References
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Wójcik, M., 2017. Selected secondary metabolites in Echium vulgare L. populations from
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Kováčik, J., Grúz, J., Klejdus, B., Štork, F., Hedbavny, J., 2012. Accumulation of metals and
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selected nutritional parameters in the field-grown chamomile anthodia. Food Chem. 131,
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Kováčik, J., Babula, P., Hedbavny, J., Švec, P., 2014a. Manganese-induced oxidative stress in
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two ontogenetic stages of chamomile and amelioration by nitric oxide. Plant Sci. 215-216,
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essential and toxic element for plants: transport, accumulation and resistance mechanisms.
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metalliferous and non-metalliferous sites: a field study. Ecotoxicology 22, 1422-1434.
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Najeeb, U., Xu, L., Ali, S.;, Jilani, G., Gong, H.J., Shen, W.Q., Zhou, W.J. 2009. Citric acid
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Santos, E.F., Santini, J.M.K., Paixão, A.P., Júnior, E.F., Lavres, J., Campos, M., dos Reis,
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Shi, Q., Zhu, Z., Xu, M., Qian, Q., Yu, J., 2006. Effect of excess manganese on the
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antioxidant system in Cucumis sativus L. under two light intensities. Environ. Exp. Bot.
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58, 197-205. Sowa, I., Paduch, R., Strzemski, M., Zielińska, S., Rydzik-Strzemska, E., Sawicki, J., Kocjan,
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R., Polkowski, J., Matkowski, A., Latalski, M., Wójciak-Kosior, M., 2018. Proliferative
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Vithana, M.D.K., Singh, Z., Johnson, S.K., 2018. Levels of terpenoids, mangiferin and
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phenolic acids in the pulp and peel of ripe mango fruit by pre-harvest spray application of
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FeSO4 (Fe2+) MgSO4 (Mg2+) and MnSO4 (Mn2+). Food Chem. 256, 71-76.
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Figure legends:
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Figure 1. Accumulation of manganese and selected minerals in the shoots of tetraploid
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chamomile after 20 days of cultivation in control soil (C) or in the soil with added 1000 µM
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Mn2+ (Mn). Data are means ± SDs (n = 3). ** and *** indicate significant difference at 0.01
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and 0.001 level of Student’s t-test between C and Mn treatment.
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Figure 2. Biomass production and selected biochemical parameters in the shoots of tetraploid
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chamomile after 20 days of cultivation in control soil (C) or in the soil with added 1000 µM
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Mn2+ (Mn). Data are means ± SDs (n = 3). *, ** and *** indicate significant difference at
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0.05, 0.01 and 0.001 level of Student’s t-test between C and Mn treatment. APX – ascorbate
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peroxidase, GPX – guaiacol peroxidase, CAT – catalase, GR – glutathione reductase, AsA –
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ascorbic acid, NPT – non-protein thiols. Units of enzymatic activity are nmol min-1 mg-1
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protein (APX, CAT, and GR) and µmol min-1 mg-1 protein (GPX).
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Figure 3. Fluorescence microscopy of oxidative stress in cotyledons of tetraploid chamomile
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after 20 days of cultivation in control soil (c) or in the soil with added 1000 µM Mn2+ (Mn). A
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– reactive oxygen species (stained with CellROX Deep Red Reagent, red signal), B – nitric
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oxide (stained with 2,3-diaminonaphthalene, blue signal), C – lipid peroxidation (stained with
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BODIPY 581/591 C11, greenish signal), D – hydroxyl radical/peroxynitrite (stained with
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aminophenyl fluorescein, green signal). Bar indicates 500 µm.
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Figure 4. Accumulation of manganese in diploid and tetraploid chamomile plants after 10
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days of cultivation in hydroponics with the addition of 100 or 1000 µM Mn2+. Control (C)
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contained 2.03 µM Mn2+ as micronutrient. Data are means ± SDs (n = 3). Values followed by
468
the same small or capital letter(s), are not significantly different according to Tukey’s test
469
(P<0.05). *, ** and *** indicate significant difference at 0.05, 0.01 and 0.001 level of
470
Student’s t-test between diploid and tetraploid plants in the given treatment.
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Figure 5. Accumulation of selected phenolic acids in the shoots of tetraploid chamomile after
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20 days of cultivation in control soil (C) or in the soil with added 1000 µM Mn2+ (Mn). Data
474
are means ± SDs (n = 3). *** indicates significant difference at 0.001 level of Student’s t-test
475
between C and Mn treatment.
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ACCEPTED MANUSCRIPT Figure 1
4000
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dry biomass (mg/shoot)
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CAT activity
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ACCEPTED MANUSCRIPT Figure 4
4000 a
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A** 2000
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shoot Mn (µg/g DW)
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b B* C
c 0 9000
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ACCEPTED MANUSCRIPT Figure 5
200 protocatechuic vanillic chlorogenic
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p-hydroxybenzoic caffeic p-coumaric
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ACCEPTED MANUSCRIPT
11.9 ± 1.71 a 1.58 ± 0.23 b 1.55 ± 0.37 b 25.5 ± 4.17 a 9.16 ± 1.01 a 20.8 ± 2.79 a
11.0 ± 1.73 a 3.70 ± 0.52 a 8.25 ± 0.87 a 25.7 ± 2.91 a 11.0 ± 2.32 a 15.7 ± 3.63 a
roots (cell wall-bound) caffeic acid p-coumaric acid ferulic acid
410.9 ± 22.1 a 93.5 ± 9.32 a 155.7 ± 16.1 a
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roots protocatechuic acid vanillic acid p-hydroxybenzoic acid chlorogenic acid caffeic acid p-coumaric acid
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100 µM Mn 28.0 ± 5.07 a 194.6 ± 13.2 b 5.47 ± 0.65 a 57.0 ± 5.83 b 4.73 ± 0.32 a 16.3 ± 2.65 a
tetraploid 1000 µM Mn C 27.1 ± 4.42 a 24.6 ± 3.24 A 236.7 ± 6.79 a 190.4 ± 15.3 A 4.84 ± 0.51 a 6.02 ± 0.23 A 86.0 ± 8.93 a 53.7 ± 4.36 B 2.41 ± 0.36 b 4.71 ± 0.59 A 18.0 ± 2.87 a 13.4 ± 2.18 A
11.8 ± 1.99 a 3.83 ± 0.19 a 8.91 ± 0.68 a 32.8 ± 3.68 a 11.4 ± 1.38 a 15.3 ± 3.26 a
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shoots protocatechuic acid vanillic acid p-hydroxybenzoic acid chlorogenic acid caffeic acid p-coumaric acid
diploid C 27.7 ± 3.18 a 199.5 ± 18.8 ab 5.43 ± 0.46 a 50.6 ± 4.78 b 4.90 ± 0.72 a 13.5 ± 2.18 a
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Table 1. Accumulation of selected phenolic acids (µg/g DW) in diploid and tetraploid chamomile plants after 10 days of cultivation in hydroponics with the addition of 100 or 1000 µM Mn2+. Control (C) contained 2.03 µM Mn2+ as micronutrient. Data are means ± SDs (n = 3). Values within rows, followed by the same small or capital letter(s), are not significantly different according to Tukey’s test (P<0.05). *, ** and *** indicate significant difference at 0.05, 0.01 and 0.001 level of Student’s t-test between diploid and tetraploid plants in the given treatment. Free phenolic acids in shoots and roots were assayed in 80% methanol extracts but cell wall-bound acids were extracted by alkaline hydrolysis of methanol-insoluble root residue.
409.3 ± 21.9 a 95.3 ± 8.02 a 111.6 ± 22.0 b
7.67 ± 0.36 A* 1.74 ± 0.34 C 1.46 ± 0.39 B 25.3 ± 3.72 B 9.13 ± 2.44 A 10.4 ± 1.85 A**
263.2 ± 34.3 b 435.2 ± 19.1 A 57.6 ± 6.03 b 63.3 ± 6.77 A* 94.3 ± 12.9 b 153.9 ± 35.4 A
26
100 µM Mn 28.2 ± 2.82 A 160.3 ± 19.1 A 6.13 ± 0.28 A 101.3 ± 14.6 A** 4.53 ± 0.37 A 14.0 ± 2.35 A
1000 µM Mn 26.0 ± 3.05 A 151.9 ± 15.0 A*** 6.24 ± 0.53 A* 103.9 ± 12.7 A 3.55 ± 0.47 A* 19.7 ± 3.17 A
7.08 ± 0.64 A* 2.78 ± 0.21 B* 14.7 ± 2.44 A* 41.2 ± 3.98 A** 10.5 ± 1.84 A 11.3 ± 1.06 A
7.62 ± 0.72 A* 4.47 ± 0.43 A 15.4 ± 1.30 A** 42.3 ± 3.94 A* 9.47 ± 0.69 A 10.7 ± 1.91 A
369.4 ± 29.4 B 66.0 ± 5.65 A** 128.7 ± 16.2 A
323.2 ± 14.5 B* 65.8 ± 5.24 A 122.4 ± 17.7 A
ACCEPTED MANUSCRIPT Research highlights
► soil Mn excess induced oxidative stress and depleted growth, thiols and ascorbate ► soil Mn excess rather depleted total soluble phenols and phenolic acids
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► shoot Mn content reached over 2800 µg/g DW with BAF 51.0 in the soil culture ► tetraploids contained less Mn in shoots and roots than diploids in hydroponics
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► phenols (mainly chlorogenic acid) were more elevated in tetraploids in hydroponics
ACCEPTED MANUSCRIPT Contribution Experimental design, spectrophotometry and manuscript preparation (JK and JH), analyses of
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minerals and phenolic acids (SD and MWK), fluorescence microscopy (PB).
S0981-9428(18)30477-7
DOI:
https://doi.org/10.1016/j.plaphy.2018.10.031
Reference:
PLAPHY 5473
To appear in:
Plant Physiology and Biochemistry
Received Date: 2 September 2018 Revised Date:
28 October 2018
Accepted Date: 28 October 2018
Please cite this article as: J. Kováčik, Sł. Dresler, M. Wójciak-Kosior, J. Hladký, P. Babula, Metabolic changes induced by manganese in chamomile, Plant Physiology et Biochemistry (2018), doi: https:// doi.org/10.1016/j.plaphy.2018.10.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Metabolic changes induced by manganese in chamomile
2 3
Jozef Kováčik
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Babula e
a*
, Sławomir Dresler b, Magdalena Wójciak-Kosior c, Juraj Hladký d, Petr
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a
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Republic
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b
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033 Lublin, Poland
Department of Biology, University of Trnava, Priemyselná 4, 918 43 Trnava, Slovak
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Department of Plant Physiology, Maria Curie-Skłodowska University, Akademicka 19, 20-
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c
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Lublin, Poland
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d
Faculty of Education, University of Trnava, Priemyselná 4, 918 43 Trnava, Slovak Republic
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e
Department of Physiology, Faculty of Medicine, Masaryk University, Kamenice 753/5, 625
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00 Brno, Czech Republic
*
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corresponding author e-mail: [email protected]
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Department of Analytical Chemistry, Medical University of Lublin, Chodźki 4a, 20-093
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ACCEPTED MANUSCRIPT ABSTRACT
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Manganese (Mn) uptake and toxicity in chamomile (Matricaria chamomilla) and changes of
20
phenolic metabolites in plants grown in the soil (1000 µM Mn2+) or hydroponic culture (100
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or 1000 µM Mn2+) were studied. Under soil cultivation, Mn excess reduced growth and
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induced symptoms of oxidative stress (including total ROS, hydroxyl radical and lipid
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peroxidation as detected by fluorescence microscopy), concomitantly with depletion of non-
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protein thiols and ascorbic acid. Total soluble phenols and individual phenolic acids were
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rather depleted (p-coumaric, chlorogenic, and protocatechuic acids) or unaltered (vanillic and
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caffeic acids). Shoot Mn content reached 2806 µg/g DW with BAF 51.0 in the soil culture. In
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hydroponics, tetraploid plants contained less Mn in both shoots and roots than diploid ones
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with bioaccumulation factor and translocation factor (diploid/tetraploid) 57.1/37.9 and
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0.39/0.32 in 1000 µM Mn treatment. Plants cultured in hydroponics revealed stimulation of
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some phenolic acids, mainly chlorogenic acid in the shoots and p-hydroxybenzoic and vanillic
31
acids in the roots (more extensively in tetraploid ones which contained less Mn). Data
32
indicate that excessive Mn accumulation has negative impact not only on the growth but also
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on phenolic metabolites in young plants mainly. Detailed comparison of the observed
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metabolic changes with limited literature focused on Mn physiology is provided as well.
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Keywords: antioxidants; fluorescence microscopy; heavy metals; soil pollution.
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Manganese (Mn) is an essential plant micronutrient which is a component of e.g. numerous
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enzymes (Millaleo et al., 2010). It naturally occurs in the soil but its elevated levels are
41
detectable in polluted soils (Kandziora-Ciupa et al., 2013). Its uptake is relatively fast process
42
in various crop or weed species (Kováčik et al., 2014b; Inostroza-Blancheteau et al., 2017)
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and is typically higher compared to other metals including also data from chamomile
44
(Matricaria chamomilla; Kováčik et al., 2010 and 2014a). As in the case of other metals, Mn
45
toxicity is variable even in genotypes of the given species with subsequent variability in the
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growth and metabolic responses (Dziwornu et al., 2018). Clear excluders or Mn accumulators
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are therefore known with translocation factor over or below 1 (Kováčik et al., 2014a;
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Inostroza-Blancheteau et al., 2017).
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Toxicity of metal excess is typically evoked by elevated reactive oxygen species
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(ROS) formation which is relatively mild under Mn excess if assayed by standard
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spectrophotometry (i.e. low increase in hydrogen peroxide or MDA content) as observed in
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several species (Farzadfar et al., 2017; Nazari et al., 2017). On the contrary, fluorescence
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microscopy as a more sensitive technique for oxidative stress detection (Kováčik and Babula,
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2017) revealed considerable enhancement of ROS and lipid peroxidation in various plants
55
(Kováčik et al., 2014a and 2014b). This metal-induced oxidative stress must be regulated by
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various mechanisms to prevent damage and eventual lethal impact on the growth. They
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include both antioxidative enzymes and non-enzymatic antioxidants. Enzymatic activities are
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typically elevated (Gangwar et al., 2010; Santos et al., 2017) while non-enzymatic
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antioxidants such as ascorbic acid (AsA) and non-protein thiols including glutathione are
60
rather depleted by Mn excess in plants (Shi et al., 2006; Gangwar et al., 2010). Phenolic
61
metabolites as widespread secondary plant products were relatively rarely been monitored
62
under Mn excess. Total soluble phenols show rather slight elevation under moderate Mn
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excess (Farzadfar et al., 2017) and more pronounced elevation under higher Mn doses
64
(Inostroza-Blancheteau et al., 2017). At the level of individual metabolites, phenolic acids
65
revealed rather negligible changes in the shoot tissue (Farzadfar et al., 2017) or in fruits
66
(Vithana et al., 2018). Chamomile is a widely used medicinal plant showing often tolerance to higher metal
68
concentrations and genotype-related pattern of the accumulation of some metals (Kováčik et
69
al., 2010 and 2012). Previous work revealed higher sensitivity of chamomile seedlings (in
70
comparison with older plants cultured in hydroponics) to Mn excess at the level of growth,
71
while symptoms of oxidative stress were rather similar (Kováčik et al., 2014a). No metabolic
72
responses were monitored in the previous studies and we therefore compared Mn excess in
73
the soil and in hydroponics in terms of Mn accumulation and the Mn impact on phenolic acids
74
in chamomile. In addition, ROS appearance and comparison of genotypes were monitored.
75
Data are explanatively compared with related studies in terms of similarities and differences
76
of Mn-induced metabolic changes.
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2. Materials and methods
79
2.1. Cultivation, experimental design and statistics
80
Soil experiment was performed in Eutric Cambisol (with sandy loamy texture) containing
81
water-soluble Mn ca. 1.50 mg/kg (Kováčik et al., 2014b) marked as control soil in the present
82
study. Parallel identical soil was enriched with Mn (as MnCl2.4H2O) to achieve 1000 µM Mn
83
(55 mg Mn/kg), marked as Mn soil in results. This concentration is ca. 1/5 from water-soluble
84
Mn content in naturally-contaminated Mn soil which evoked strong growth depression of
85
several crops in the previous work (Kováčik et al., 2014b). Soil moisture was maintained at
86
60% of water holding capacity with distilled water and no additional nutrients were applied
87
(pot diameter 10 cm with 0.5 kg of the soil). Cultivation was realized under laboratory
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89
tubes L36W/840 (Lumilux, Osram), 25/20°C day/night temperature and relative humidity of
90
~60 % (Kováčik et al., 2014a). Tetraploid chamomile (Matricaria chamomilla cv. Lutea,
91
Asteraceae) seeds were sown directly on the surface of the soil (20 seeds per pot, 20 pots for
92
each treatment) and germinated within 48 – 72 h. Seedlings were cultured for additional 20
93
days when the growth responses between control and Mn-enriched soil became visible.
94
Owing to tiny roots, only above-ground biomass was analyzed for minerals and biochemical
95
parameters. Seedlings from one pot were pooled to achieve enough biomass for individual
96
measurements. For fresh mass-requiring parameters, samples were extracted with cold mortar
97
and pestle as mentioned below and dry samples (dried at 75°C to constant weight) were
98
analyzed for mineral nutrients and phenols.
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In the subsequent experiment, diploid and tetraploid chamomile plants were pre-
100
cultured in the sand followed by 4 weeks of cultivation in hydroponics with Hoagland
101
solution (Kováčik et al., 2014a). Thereafter, plants were exposed to 100 or 1000 µM Mn (as
102
MnCl2.4H2O), control contained 2.03 µM Mn2+ as micronutrient and pH was checked to be
103
6.0 in all treatments. One 2-L box containing 10 plants was used for each treatment with two
104
repetitions, thus the whole experiment included 12 boxes. After 10 days of cultivation with
105
Mn excess, shoots and roots were separated, roots washed trice with deionised water and
106
dried (at 75°C to constant weight) for the assay of Mn accumulation and phenolic metabolites.
107
Student’s t-test was used to compare the differences between control and Mn-enriched
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soil. Data from hydroponics were evaluated using ANOVA followed by a Tukey’s test
109
(MINITAB Release 11, Minitab Inc.; State College, Pennsylvania) at P<0.05 for diploid or
110
tetraploid samples individually while Student’s t-test was used to determine difference
111
between diploid and tetraploid samples in the given treatment.
112
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114
Samples were prepared by mineralization of dry plant material (50 mg) in the mixture of
115
concentrated HNO3 and water (5 + 5 mL) using microwave decomposition (Ethos Sel
116
Microwave Extraction Labstation, Milestone Inc.) at 200°C over 1 h (complete duration of the
117
mineralization program). Resulting clear solution was placed to inert plastic flasks and diluted
118
to a final volume of 20 mL. All measurements were carried out using an atomic absorption
119
spectrometer (Polarised Zeeman Z – 8200, Hitachi, Tokyo, Japan) as reported previously
120
(Kováčik et al., 2014a; Dresler et al., 2017).
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2.3. Assay of enzymatic and non-enzymatic antioxidants
123
For the assay of enzymatic activities, whole shoots were homogenized in potassium
124
phosphate buffer containing 1% insoluble PVPP (pH 7.0) using cold mortar and pestle with
125
the addition of small amount of inert so-called sea sand (Penta s. r. o., Prague, Czech
126
Republic) to achieve complete tissue disruption, followed by centrifugation at 14 000 g for 15
127
min at 4°C. Soluble proteins were quantified according to Bradford method and bovine serum
128
albumin as standard (595 nm) as reported previously (Kováčik et al., 2014b). Ascorbate
129
peroxidase (APX) and guaiacol peroxidase (GPX) activities were measured as the oxidation
130
of ascorbate and guaiacol at 290 and 470 nm, respectively; glutathione reductase (GR)
131
activity was assayed as the reduction of GSSG at 412 nm and catalase (CAT) activity as the
132
reduction of H2O2 to water at 240 nm (Kováčik et al., 2014a and 2014b). Randomly selected
133
supernatants were boiled and assayed in order to check that reactions were enzymatic.
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For the assay of ascorbic acid (AsA) and non-protein thiols (NPT), samples were
135
extracted in 0.1 M HCl (0.1 g FW/mL) using cold mortar and pestle followed by
136
centrifugation
as
mentioned
above.
Reduced
6
AsA
content
was
determined
by
ACCEPTED MANUSCRIPT 137
bathophenanthroline method and NPT by 5,5ʼ-dithiobis-(2-nitrobenzoic acid) as reported in
138
detail previously (Kováčik et al., 2017).
139 2.4. Fluorescence microscopy
141
For microscopic analyses, cotyledons (the oldest photosynthetic part of the shoot) were used.
142
Whole individual fresh cotyledons were immediately stained and observed. General
143
accumulation of ROS was monitored using widely-used CellROX® Deep Red Reagent
144
(644Ex/665Em
145
radical/peroxynitrite reagent (aminophenyl fluorescein, 490Ex/515Em nm, Sigma-Aldrich).
146
Nitric oxide or reactive nitrogen species (RNS) were visualized with 2,3-diaminonaphthalene
147
(DAN, 365Ex/415Em nm, Sigma-Aldrich) and lipid peroxidation by BODIPY® 581/591 C11
148
lipid peroxidation sensor (581Ex/591Em nm, Life Technologies, USA). Staining procedures
149
were conducted as reported previously (Kováčik et al., 2014a; Kováčik and Babula, 2017).
150
Staining solution was always removed by respective buffer and samples were observed using
151
fluorescence microscope (Axioscop 40, Zeiss, Germany) and appropriate set of filters.
Life
Technologies,
USA)
and
specifically
with
hydroxyl
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2.5. Assay of phenolic metabolites
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Total soluble phenols were extracted with 80% methanol (100 mg DW/mL) and quantified
155
using Folin-Ciocalteu method with gallic acid as standard and detection at 750 nm (Kováčik
156
et al., 2014b).
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For the assay of individual phenolic acids, methanol extracts mentioned above were
158
extracted twice with anhydrous diethyl ether, evaporated to dryness using SpeedVac and
159
dissolved in 0.1 mL of 80% aqueous methanol. Root cell wall-bound phenols were measured
160
after alkaline hydrolysis of methanol-insoluble root residue: samples were washed trice with
161
methanol until no soluble phenols were detectable. Then they were treated with 1 M NaOH
7
ACCEPTED MANUSCRIPT and heated for 90 min at 60°C to release cell wall-bound phenols, acidified, extracted twice
163
with diethyl ether, evaporated to dryness and dry residue was dissolved in 80% methanol as
164
mentioned above (Kováčik et al., 2011). Quantification was done by HPLC/CE system and
165
identification by comparison of the retention time and absorption spectrum similarity between
166
the standards (all from Sigma-Aldrich) and the samples for the detectable phenolic acids as
167
reported previously (Dresler et al., 2017; Sowa et al., 2018).
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3. Results and discussion
170
3.1. Impact of soil Mn on its accumulation and mineral nutrients
171
Water-soluble Mn content in control soil (ca. 1.50 mg/kg = µg/g) is similar to earlier study
172
from Poland (1.70 mg Mn/kg; Nadgórska-Socha et al., 2013) but lower compared to other
173
Polish sites (average value 4.0 – 9.7 mg Mn/kg; Kandziora-Ciupa et al., 2013). Under these
174
conditions, control chamomile shoots contained 62.4 µg Mn/g DW which is similar to
175
previous work where several crops contained 53 – 66 µg Mn/g DW in control soil (Kováčik et
176
al., 2014b). Addition of Mn to soil (55 mg/kg) led to shoot Mn content over 2800 µg/g DW
177
(Fig. 1). This bioavailable Mn content is higher compared to “naturally” polluted localities in
178
Poland where 3.54 – 24.89 mg Mn/kg soil was reported (Kandziora-Ciupa et al., 2013): under
179
these conditions, Vaccinium leaves contained 38.8 – 398 µg Mn/g DW, which is far lower
180
compared to our present data. On the contrary, water-soluble soil Mn content 280 mg/kg in an
181
earlier study led to shoot Mn amount 2300 – 13 600 µg/g DW in various crops (Kováčik et
182
al., 2014b). In rice, higher Mn availability in the soil led to higher shoot Mn content (up to ca.
183
4000 µg/g DW; Dziwornu et al., 2018).
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Owing to variability in soil and tissue Mn content, bioaccumulation factor is more
185
suitable for comparison between studies. Shoot bioaccumulation factor (BAF) is defined as
186
the ratio of shoot Mn content (µg/g DW) to soil Mn content (µg/g DW), i.e. 1.50 (control soil)
8
ACCEPTED MANUSCRIPT and 55 mg Mn/kg (Mn soil) in this case. The values are 41.6 (control) and 51.0 (Mn excess)
188
for chamomile and are typically higher than those observed in various crops exposed to Mn
189
excess: 8.4 – 48.9 (using shoot Mn content/water-soluble soil Mn content; Kováčik et al.,
190
2014b). Few other studies also reported Mn uptake in plants from natural localities and shoot
191
Mn BAF can be calculated. For example, shoot BAF in Vaccinium myrtillus (considering
192
potentially bioavailable soil Mn fraction extracted with 0.01 M CaCl2) was lower in the soil
193
from polluted localities (average value 17 for various time of harvest) in comparison with
194
control localities (average value 140 for various time of harvest; Kandziora-Ciupa et al.,
195
2013). On the contrary, Plantago lanceolata and Cardaminopsis arenosa from polluted
196
locality (mining and metallurgic activity, bioavailable Mn was only up 3-times higher in
197
polluted soil), revealed similar shoot BAF if compared to control site (average value 4.35 in
198
P. lanceolata polluted sites vs. 3.6 in control; average value 11.8 in C. arenosa polluted sites
199
vs. 14 in control; Nadgórska-Socha et al., 2013). All these data confirm that various plant
200
species have various trends of Mn accumulation and that chamomile (considering typically
201
higher shoot BAF both in control and Mn soil) has higher Mn accumulation potential in
202
comparison with mentioned species.
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Excess of Mn, as in the case of excess of other essential or non-essential metallic ions,
204
affects sorption of various nutrients. It was surprising to find that K and Zn content remained
205
unaltered by excessive Mn accumulation (Fig. 1) as previously observed in hydroponically
206
cultured chamomile (Kováčik et al., 2014a) and partially in some crops cultured in naturally-
207
contaminated Mn soil (Kováčik et al., 2014b). In agreement, soybean exposed to 100 – 300
208
µM Mn showed slight or no change in K, Ca, Mg, Zn and Cu content; interestingly,
209
movement of Ca from the healthy area to necrotic area (evoked by Mn excess) and the
210
opposite pattern of K movement were reported (Santos et al., 2017). On the contrary,
211
chamomile in the present study showed depletion of Ca and Mg amount (Fig. 1) as previously
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213
indicating that responses are evoked rather by applied Mn concentration than by the mode of
214
cultivation. Strong depletion of Ca and Mg content has also been found in crops cultured in
215
naturally-contaminated Mn soil (Kováčik et al., 2014b) and it seems that Mn excess is a
216
strong competitor of divalent cations (mainly Ca and Mg). In agreement with this assumption,
217
Mn-induced (increase from 9.1 µM in control to 150 µM Mn) depletion of Mg in Tanacetum
218
parthenium was reversed by an increase in exogenous Mg dose from 1 mM (= 1000 µM) in
219
control to 2 – 4 mM and Mn accumulation decreased with increasing Mg dose (Farzadfar et
220
al., 2017). Amount of cadmium, an example of non-essential and toxic metal, was lower in
221
control chamomile (Fig. 1) than in the previously tested crops (Kováčik et al., 2014b) and Mn
222
excess stimulated Cd accumulation.
223
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3.2. Impact of soil Mn on oxidative stress and antioxidants
225
Mn-induced depression of the growth and soluble proteins of chamomile (Fig. 2) was also
226
previously observed in the crops cultured in the soil with 280 mg water-soluble Mn/kg
227
(Kováčik et al., 2014b). Other species are yet more sensitive to Mn excess such as Pisum
228
sativum cultured with 100 – 250 µM Mn (Gangwar et al., 2010) or soybean cultured with 10 –
229
300 µM Mn (Santos et al., 2017) while grass Lolium perenne (accumulating Mn considerably)
230
was resistant to even 750 µM Mn (Inostroza-Blancheteau et al., 2017).
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Sensitivity of the given species reflected in the growth depression could be evoked,
232
among other, by changes in the ROS balance (leading to damage of essential biomolecules if
233
excessive ROS formation is not effectively controlled). In agreement, depletion of APX and
234
GR activities may be related to depleted accumulation of ascorbic acid and non-protein thiols
235
in response to Mn excess (Fig. 2). On the contrary, GPX and CAT activities were stimulated
236
by Mn excess, as previously observed in crops cultured in naturally-contaminated Mn soil
10
ACCEPTED MANUSCRIPT (Kováčik et al., 2014b). In agreement with our observations, Mn excess (600 µM) variously
238
affected CAT, APX, GPX and GR in cucumber (Shi et al., 2006) or in pea cultured with 100 –
239
250 µM Mn (Gangwar et al., 2010). On the contrary, all antioxidative enzymes showed
240
elevation in response to 10 – 300 µM Mn in soybean but it is questionable whether the unit of
241
activity at the level of mmol/min/mg protein is realistic (Santos et al., 2017). These data
242
indicate that antioxidative enzymes are variously affected by Mn excess, leading to alteration
243
of ROS balance. Fluorescence microscopy confirmed considerable elevation of both general
244
ROS and hydroxyl radical in response to Mn, concomitantly with elevated lipid peroxidation
245
and nitric oxide signal (Fig. 3). All these responses are similar to those observed in
246
chamomile cultured in hydroponics with 100 – 1000 µM Mn (Kováčik et al., 2014a) and
247
confirm responses of oxidative symptoms to Mn excess independently on the ontogenetic
248
stage. In agreement, crops cultured in the soil with water-soluble Mn 280 mg/kg (ca. 5 mM)
249
showed considerably enhanced ROS, NO and lipid peroxidation signal which may be a reason
250
for suppressed growth (Kováčik et al., 2014b). On the contrary to our qualitative data
251
showing strong enhancement of oxidative stress symptoms under Mn excess, quantitative data
252
revealed significant but rather mild increase in H2O2 and superoxide accumulation in
253
Tanacetum parthenium exposed to 150 µM Mn (Farzadfar et al., 2017) or in Mentha aquatica
254
treated by 160 µM Mn (Nazari et al., 2017). In addition, only slight increase in lipid
255
peroxidation has been observed in rice (we note that unit of MDA content nmol/mL/g is
256
unclear; Dziwornu et al., 2018) and mainly Mn dose 750 µM elevated strongly MDA level in
257
Lolium
258
spectrophotometry is common in the literature, it does not reach sensitivity of the
259
fluorescence microscopy to detect slight changes in the ROS formation (see Kováčik and
260
Babula, 2017 for details).
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perenne
(Inostroza-Blancheteau
et
11
al.,
2017).
Overall,
though
standard
ACCEPTED MANUSCRIPT Depletion of ascorbic acid and non-protein thiols in response to Mn excess could be a
262
reason for enhancement of oxidative stress appearance (cf. Figs 2 and 3) and growth
263
inhibition as mentioned above and the same was previously observed in the cucumber
264
exposed to 600 µM Mn (Shi et al., 2006) or in the pea plants treated by 100 – 250 µM Mn
265
(Gangwar et al., 2010). Data from field-collected plants growing on the soil with various
266
metallic contaminations including higher Mn amount showed variously affected glutathione
267
and non-protein thiol contents: Mn revealed negative correlation with GSH in Vaccinium
268
myrtillus (Kandziora-Ciupa et al., 2013) or in Cardaminopsis arenosa (Nadgórska-Socha et
269
al., 2013) while non-protein thiols did not. We note, however, that GSH content in the two
270
mentioned species cannot reach up to 250 µmol/g FW (= over 760 mg/g DW considering
271
tissue water content 90%) and is in strong contradiction to non-protein thiol content up to 1.8
272
µmol/g FW (as GSH is one of them). It is concluded that management of thiols and ascorbate
273
accumulation in plants, to prevent their depletion, could provide a tool for better growth
274
performance under Mn excess.
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3.3. Impact of solution Mn on its accumulation
277
We note that applied Mn doses did not suppress growth of hydroponically cultured plants
278
owing to longer pre-cultivation prior to Mn application while seedlings are more sensitive as
279
reported previously (Kováčik et al., 2014a) and in this work (Fig. 2). Assay of Mn content in
280
plants cultured in hydroponics revealed preferential Mn accumulation in the roots compared
281
to shoots except for control (Fig. 4). Ten fold increase in exogenous Mn (from 100 to 1000
282
µM) evoked ca. 5-6-fold and ca. 4-fold increase in shoot and root Mn accumulation,
283
respectively. These data indicate that chamomile readily absorbs Mn to shoots (over 400 µg
284
Mn/g DW at 100 µM Mn) in comparison with e.g. soybean (ca. 200 µg Mn/g DW at 100 µM
285
Mn; Santos et al., 2017), Juncus effusus (137 µg Mn/g DW at 100 µM Mn; Najeeb et al.,
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ACCEPTED MANUSCRIPT 2009) or Mentha aquatica (193 µg Mn/g DW at 160 µM Mn; Nazari et al., 2017). On the
287
contrary, related species Tanacetum parthenium (Asteraceae family too) contained more Mn
288
in shoots (1898 µg Mn/g DW at 150 µM Mn with normal/1 mM Mg in the solution; Farzadfar
289
et al., 2017) and grass species Lolium perenne revealed the same (842 – 938 µg Mn/g DW at
290
150 µM Mn; Inostroza-Blancheteau et al., 2017). It was observed that tetraploid chamomile
291
contained less Mn in both shoots and roots than diploid ones under Mn excess (Fig. 4). In
292
agreement, inflorescences of field-grown tetraploid chamomile contained less Cd, Hg, Cr, Cu,
293
Al, Mg, Ca and K than diploid flowers (Kováčik et al., 2012) and lower Cd but not Ni
294
accumulation was observed in chamomile vegetative tissue (Kováčik et al., 2010). Ploidy
295
level of chamomile may therefore affect accumulation of various nutrients which could be
296
usable in selection of cultivars with lower metallic contamination (for pharmaceutical
297
purposes) or with higher metal accumulation (for remediation purposes).
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As mentioned above, shoot BAF values are also calculable for solution culture as
299
shoot Mn (µg/g DW)/solution Mn (µg/mL) ratio (from the Fig. 4, diploid/tetraploid plants):
300
21.1/23.4 (control), 85.8/73.1 (100 µM Mn treatment) and 57.1/37.9 (1000 µM Mn
301
treatment). Value in tetraploid shoots in 1000 µM Mn treatment (37.9) is lower than in soil-
302
cultured plants (51.0, see above) but is not considerably lower considering totally different
303
mode of cultivation. It is wort noting that lower BAF values also reflect lower Mn content in
304
tetraploid shoots (in comparison with diploid ones) and ploidy-dependent pattern is therefore
305
visible as mentioned above. For comparison, shoot BAF value of Mn in soybean cultured with
306
100 µM Mn is lower (36, calculable from the data by Santos et al., 2017) and reflects lower
307
shoot Mn content too.
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On the contrary to BAF which reflects tissue metal content in relation to exogenously
309
applied metal dose, translocation factor (TF) is a relative unit of shoot metal (µg/g DW) to
310
root metal content (µg/g DW) ratio and indicates tendency of root to shoot metal movement.
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ACCEPTED MANUSCRIPT In chamomile, TF values (calculable from the Fig. 4) are (diploid/tetraploid plants) 2.1/2.6
312
(control), 0.21/0.26 (100 µM Mn treatment) and 0.39/0.32 (1000 µM Mn treatment). These
313
data are in agreement with previous report from tetraploid plants cultured in hydroponics (TF
314
0.31 and 0.34 in 100 and 1000 µM Mn treatment) while seedling cultured with water Mn
315
solution showed higher TF (0.57 and 0.47 in 100 and 1000 µM Mn treatment; Kováčik et al.,
316
2014a). Surprisingly, species with lower shoot Mn content in comparison with chamomile
317
may have higher TF such as Juncus effusus (TF 0.73 at 100 µM Mn; Najeeb et al., 2009) or
318
soybean (TF>1 at 100 – 300 µM Mn; Santos et al., 2017), confirming excluder character of
319
chamomile (i.e. preferential Mn accumulation in the roots). However, chamomile could be
320
more suitable for eventual accumulation (in the shoots) and phytostabilization (root
321
accumulation) of Mn ions.
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322
3.4. Impact of soil vs. solution Mn on phenols
324
Phenolic metabolites are widespread secondary plant products involved also in responses to
325
metallic stress as previously observed using specific inhibitor just in chamomile (Kováčik et
326
al., 2011). Slight but significant depletion of total soluble phenols (common abbreviation
327
TPC) in the shoots of plants cultured in Mn soil (Fig. 2) indicates negative impact of Mn
328
excess specifically in chamomile because higher soil Mn dose in the previous study did not
329
deplete soluble phenols in several crops (Kováčik et al., 2014b). In agreement, TPC content
330
was rather elevated by Mn excess 50 – 150 µM in Tanacetum parthenium (Farzadfar et al.,
331
2017) or by higher Mn doses (350 or 750 µM) in Lolium perenne (Inostroza-Blancheteau et
332
al., 2017). In the fruits, exogenous application of 0.2 or 0.3% Mn2+ had negligible impact on
333
TPC in mango (Vithana et al., 2018). Subsequent assay of individual phenolic acids in
334
chamomile shoots revealed their unaltered or depleted accumulation (as TPC also did), only
335
p-hydroxybenzoic acid increased (Fig. 5). Is seems that Mn-induced oxidative stress in soil-
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ACCEPTED MANUSCRIPT 336
cultured plants was strong enough to suppress not only general antioxidants (ascorbate and
337
thiols) but also phenolic metabolites. In the hydroponically-cultured plants, chlorogenic acid was the only compound
339
significantly enhanced by Mn excess both in diploid and tetraploid shoots while other acids
340
remained unaffected (Table 1). In the roots (free fraction), p-hydroxybenzoic and vanillic
341
acids increased in both genotypes while chlorogenic acid in tetraploid roots only (Table 1).
342
Various responses of chamomile genotypes at the level of phenolic metabolites have been
343
previously reported (Kováčik et al., 2010). Chlorogenic acid is an important antioxidant
344
which is often elevated by various stressors including metals such as Cd in chamomile
345
(Kováčik et al., 2011) and its elevation could indicate the same function under Mn excess too.
346
However, stimulation by Mn excess was lower in comparison with other metals. In
347
agreement, only gallic and ferulic acids increased in response to 50 or 150 µM Mn in
348
Tanacetum parthenium while other phenolic acids (p-hydroxybenzoic, salicylic, caffeic and
349
chlorogenic acids) remained unaltered (Farzadfar et al., 2017): it is wort noting that many
350
acids increased after application of elevated Mg dose which suppressed Mn uptake, thus
351
probably preventing toxic impact of Mn on phenolic biosynthesis. This is indirectly
352
confirmed by our present data because plants cultured in the soil revealed growth retardation
353
and depleted Mg content (Figs 1 and 2). Cell wall-bound acids were rather depleted under Mn
354
excess and more extensively in diploid roots, indicating genotype-related differences (Table
355
1). Significance of this phenomenon remains to be elucidated but it was previously observed
356
also under Cd excess (Kováčik et al., 2011).
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357 358
Conclusions
359
Present study showed that the growth retardation induced by soil Mn excess is related to the
360
appearance of oxidative stress and depletion of antioxidants (not only ascorbic acid and thiols
15
ACCEPTED MANUSCRIPT but also phenolic metabolites) with shoot Mn bioaccumulation factor 51.0. In hydroponics,
362
tetraploid shoots in 1000 µM Mn treatment showed BAF 37.9 which is lower than in soil-
363
cultured plants, but is not considerably low considering totally different mode of cultivation.
364
Higher accumulation of Mn in both shoots and roots of diploid plants has also been recorded.
365
Since no growth retardation was found, plants cultured in hydroponics revealed stimulation of
366
some phenolic acids, mainly chlorogenic acid in the shoots and p-hydroxybenzoic and vanillic
367
acids in the roots. It seems that excessive Mn accumulation has rather negative impact on
368
phenolic metabolites in young plants mainly.
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369 Acknowledgement
371
The work was supported by Slovak grant agency VEGA (project no. 1/0041/18).
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372 Disclosure statement
374
The authors declare that there are no conflicts of interest.
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375
Role of the funding source
377
Sponsor had no involvement in the present study.
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376
378 Contribution
380
Experimental design, spectrophotometry and manuscript preparation (JK and JH), analyses of
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References
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Dresler, S., Rutkowska , E., Bednarek, W., Stanisławski, G., Kubrak, T., Bogucka-Kocka, A.,
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Wójcik, M., 2017. Selected secondary metabolites in Echium vulgare L. populations from
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Kováčik, J., Grúz, J., Klejdus, B., Štork, F., Hedbavny, J., 2012. Accumulation of metals and
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Kováčik, J., Babula, P., Hedbavny, J., Švec, P., 2014a. Manganese-induced oxidative stress in
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two ontogenetic stages of chamomile and amelioration by nitric oxide. Plant Sci. 215-216,
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contaminated manganese soil to selected crops. J. Agric. Food Chem. 62, 7287-7296.
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Kováčik, J., Štěrbová, D., Babula, P., Švec, P., Hedbávný, J., 2014b. Toxicity of naturally-
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antioxidant system in Cucumis sativus L. under two light intensities. Environ. Exp. Bot.
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Vithana, M.D.K., Singh, Z., Johnson, S.K., 2018. Levels of terpenoids, mangiferin and
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Figure legends:
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Figure 1. Accumulation of manganese and selected minerals in the shoots of tetraploid
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chamomile after 20 days of cultivation in control soil (C) or in the soil with added 1000 µM
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Mn2+ (Mn). Data are means ± SDs (n = 3). ** and *** indicate significant difference at 0.01
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and 0.001 level of Student’s t-test between C and Mn treatment.
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Figure 2. Biomass production and selected biochemical parameters in the shoots of tetraploid
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chamomile after 20 days of cultivation in control soil (C) or in the soil with added 1000 µM
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Mn2+ (Mn). Data are means ± SDs (n = 3). *, ** and *** indicate significant difference at
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0.05, 0.01 and 0.001 level of Student’s t-test between C and Mn treatment. APX – ascorbate
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peroxidase, GPX – guaiacol peroxidase, CAT – catalase, GR – glutathione reductase, AsA –
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ascorbic acid, NPT – non-protein thiols. Units of enzymatic activity are nmol min-1 mg-1
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protein (APX, CAT, and GR) and µmol min-1 mg-1 protein (GPX).
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Figure 3. Fluorescence microscopy of oxidative stress in cotyledons of tetraploid chamomile
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after 20 days of cultivation in control soil (c) or in the soil with added 1000 µM Mn2+ (Mn). A
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– reactive oxygen species (stained with CellROX Deep Red Reagent, red signal), B – nitric
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oxide (stained with 2,3-diaminonaphthalene, blue signal), C – lipid peroxidation (stained with
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BODIPY 581/591 C11, greenish signal), D – hydroxyl radical/peroxynitrite (stained with
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aminophenyl fluorescein, green signal). Bar indicates 500 µm.
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Figure 4. Accumulation of manganese in diploid and tetraploid chamomile plants after 10
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days of cultivation in hydroponics with the addition of 100 or 1000 µM Mn2+. Control (C)
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contained 2.03 µM Mn2+ as micronutrient. Data are means ± SDs (n = 3). Values followed by
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the same small or capital letter(s), are not significantly different according to Tukey’s test
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(P<0.05). *, ** and *** indicate significant difference at 0.05, 0.01 and 0.001 level of
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Student’s t-test between diploid and tetraploid plants in the given treatment.
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Figure 5. Accumulation of selected phenolic acids in the shoots of tetraploid chamomile after
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20 days of cultivation in control soil (C) or in the soil with added 1000 µM Mn2+ (Mn). Data
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are means ± SDs (n = 3). *** indicates significant difference at 0.001 level of Student’s t-test
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between C and Mn treatment.
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11.9 ± 1.71 a 1.58 ± 0.23 b 1.55 ± 0.37 b 25.5 ± 4.17 a 9.16 ± 1.01 a 20.8 ± 2.79 a
11.0 ± 1.73 a 3.70 ± 0.52 a 8.25 ± 0.87 a 25.7 ± 2.91 a 11.0 ± 2.32 a 15.7 ± 3.63 a
roots (cell wall-bound) caffeic acid p-coumaric acid ferulic acid
410.9 ± 22.1 a 93.5 ± 9.32 a 155.7 ± 16.1 a
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roots protocatechuic acid vanillic acid p-hydroxybenzoic acid chlorogenic acid caffeic acid p-coumaric acid
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100 µM Mn 28.0 ± 5.07 a 194.6 ± 13.2 b 5.47 ± 0.65 a 57.0 ± 5.83 b 4.73 ± 0.32 a 16.3 ± 2.65 a
tetraploid 1000 µM Mn C 27.1 ± 4.42 a 24.6 ± 3.24 A 236.7 ± 6.79 a 190.4 ± 15.3 A 4.84 ± 0.51 a 6.02 ± 0.23 A 86.0 ± 8.93 a 53.7 ± 4.36 B 2.41 ± 0.36 b 4.71 ± 0.59 A 18.0 ± 2.87 a 13.4 ± 2.18 A
11.8 ± 1.99 a 3.83 ± 0.19 a 8.91 ± 0.68 a 32.8 ± 3.68 a 11.4 ± 1.38 a 15.3 ± 3.26 a
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shoots protocatechuic acid vanillic acid p-hydroxybenzoic acid chlorogenic acid caffeic acid p-coumaric acid
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Table 1. Accumulation of selected phenolic acids (µg/g DW) in diploid and tetraploid chamomile plants after 10 days of cultivation in hydroponics with the addition of 100 or 1000 µM Mn2+. Control (C) contained 2.03 µM Mn2+ as micronutrient. Data are means ± SDs (n = 3). Values within rows, followed by the same small or capital letter(s), are not significantly different according to Tukey’s test (P<0.05). *, ** and *** indicate significant difference at 0.05, 0.01 and 0.001 level of Student’s t-test between diploid and tetraploid plants in the given treatment. Free phenolic acids in shoots and roots were assayed in 80% methanol extracts but cell wall-bound acids were extracted by alkaline hydrolysis of methanol-insoluble root residue.
409.3 ± 21.9 a 95.3 ± 8.02 a 111.6 ± 22.0 b
7.67 ± 0.36 A* 1.74 ± 0.34 C 1.46 ± 0.39 B 25.3 ± 3.72 B 9.13 ± 2.44 A 10.4 ± 1.85 A**
263.2 ± 34.3 b 435.2 ± 19.1 A 57.6 ± 6.03 b 63.3 ± 6.77 A* 94.3 ± 12.9 b 153.9 ± 35.4 A
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100 µM Mn 28.2 ± 2.82 A 160.3 ± 19.1 A 6.13 ± 0.28 A 101.3 ± 14.6 A** 4.53 ± 0.37 A 14.0 ± 2.35 A
1000 µM Mn 26.0 ± 3.05 A 151.9 ± 15.0 A*** 6.24 ± 0.53 A* 103.9 ± 12.7 A 3.55 ± 0.47 A* 19.7 ± 3.17 A
7.08 ± 0.64 A* 2.78 ± 0.21 B* 14.7 ± 2.44 A* 41.2 ± 3.98 A** 10.5 ± 1.84 A 11.3 ± 1.06 A
7.62 ± 0.72 A* 4.47 ± 0.43 A 15.4 ± 1.30 A** 42.3 ± 3.94 A* 9.47 ± 0.69 A 10.7 ± 1.91 A
369.4 ± 29.4 B 66.0 ± 5.65 A** 128.7 ± 16.2 A
323.2 ± 14.5 B* 65.8 ± 5.24 A 122.4 ± 17.7 A
ACCEPTED MANUSCRIPT Research highlights
► soil Mn excess induced oxidative stress and depleted growth, thiols and ascorbate ► soil Mn excess rather depleted total soluble phenols and phenolic acids
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► shoot Mn content reached over 2800 µg/g DW with BAF 51.0 in the soil culture ► tetraploids contained less Mn in shoots and roots than diploids in hydroponics
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► phenols (mainly chlorogenic acid) were more elevated in tetraploids in hydroponics
ACCEPTED MANUSCRIPT Contribution Experimental design, spectrophotometry and manuscript preparation (JK and JH), analyses of
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minerals and phenolic acids (SD and MWK), fluorescence microscopy (PB).