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Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants
Accepted Manuscript Title: Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants Authors: Klara Andr´es Rask, Jesper Liengaard Johansen, Rasmus Kjøller, Flemming Ekelund PII: DOI: Reference:
S0098-8472(18)31901-4 https://doi.org/10.1016/j.envexpbot.2019.02.022 EEB 3710
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
21 December 2018 20 February 2019 20 February 2019
Please cite this article as: Rask KA, Johansen JL, Kjøller R, Ekelund F, Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants, Environmental and Experimental Botany (2019), https://doi.org/10.1016/j.envexpbot.2019.02.022 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.
Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants
Klara Andrés Rask, Jesper Liengaard Johansen, Rasmus Kjøller, Flemming Ekelund
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Terrestrial Ecology Section, Department of Biology, University of Copenhagen,
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Universitetsparken 15, 2100 Copenhagen Ø, Denmark
Klara Andrés Rask*: [email protected] Jesper Liengaard Johansen: [email protected] Rasmus Kjøller: [email protected]
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Flemming Ekelund: [email protected]
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*Author of correspondence
Date of submission: December 21, 2018
Date of resubmission (major revision): February 13, 2019
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Date of resubmission (minor revision): February 20, 2019
Number of figures: 6
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Image fitting: Figure 1 (2-column), Figure 2 (1-column), Figure 3 (1-column), Figure 4 (1column), Figure 5 (1-column), Figure 6 (1-column)
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Number of tables: 2 Supporting information: 0
Word count: 3341
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Graphical abstract
Highlights
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The combination of plant species and AM colonisation determines Cd uptake in plants. Especially plants with a high degree of colonisation are protected by AM through a high root retention and a low translocation to shoots. Exposure to Cd increases AM colonisation up to a certain threshold where the conditions for the symbiosis breaks down.
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Abstract
Cadmium (Cd) is one of the most toxic heavy metals found in soil. Arbuscular mycorrhiza (AM)
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is known to reduce Cd-translocation in plants by immobilising Cd in the root system. The effect of mycorrhiza on plant Cd-uptake is usually studied in simple systems with single strains
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of mycorrhizal fungi and few levels of Cd. Here we studied how a wide range of soil Cd concentrations affected plant AM-colonisation, and how the species-specific differences in
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AM-colonisation affected uptake, translocation, and toxicity of Cd in plants in a system with naturally occurring mycorrhizal fungi. Six plant species were grown in pots in a greenhouse across seven levels of Cd, which made it possible to model dose-response curves, and calculate EC50 for each plant species. We found a remarkable trend where Cd at moderate levels stimulated mycorrhizal colonisation until a certain threshold where the symbiosis breaks down. Our results support the existence of a protective effect of AM fungi against Cd,
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as the symbiosis reduces Cd-translocation to shoots, especially in plants with very high AMcolonisation. Thus, we conclude that it is the combination of plant species and AM colonisation that determines Cd uptake in plants. AM is therefore an essential trait to consider when growing plants in Cd-polluted soil.
Keywords: Ecotoxicology, heavy metals, mycorrhiza, plant-microbe interactions, bio-
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accumulation, translocation factor
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Abbreviations1
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Arbuscular mycorrhiza (AM), cadmium (Cd)
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1. Introduction Cadmium (Cd) is a heavy metal of major health and environmental concern. It is considered toxic even in small concentrations and has high mobility in food chains (Peralta-Videa et al., 2009; Thévenod and Lee, 2013). Cd pollution mostly originates from the metal industry where it is emitted during the refining process (Halada et al., 2008; Cullen and Maldonado 2013). Cd
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also occurs as a trace element in soil amendments (e.g. phosphate fertilizers, sewage sludge, and wood ash) and may thereby end up in food crops (Jiao et al., 2004; Li et al., 2016). Pollution of agricultural land is not only a problem due to the risk of human and animal consumption
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(Xiong et al., 2014; Zong et al., 2015), but also because of the negative effects Cd has on plant growth even at relatively low soil concentrations (Krämer, 2010; White and Brown, 2010).
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Arbuscular mycorrhizal (AM) fungi form a mutualistic symbiosis with more than 70 % of all
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vascular plant species (Brundrett, 2009; Smith and Read, 2010). The interaction between AM fungi and plants is not a simple relationship, but a continuum of reciprocal benefits depending
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on environmental conditions (Brundrett, 1991; Lekberg et al., 2010; Hempel et al., 2013).
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Plants have intrinsically different degrees of colonisation that vary from consistently high, to intermediate or facultative associations (Zubek and Błaszkowski, 2009; Lekberg et al., 2010;
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Moora, 2014; Brundrett, 2017). Whereas, some plants completely lack the ability to form AM (Koide and Schreiner, 1992; Brundrett, 2004, 2009).
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Mycorrhizal symbiosis increases the uptake of plant nutrients, but may also increase the uptake of non-essential metals (Galli et al., 1994; Leyval et al., 1997; Parmar et al., 2013).
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However, several studies indicate that mycorrhizal colonisation provides plants with an advantage, because toxic elements are immobilised within the mycelia or in the root system (Kaldorf et al., 1999; Joner et al., 2000; Gonzales-Chaves et al., 2004; Hildebrandt et al., 2007;
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Jiang et al., 2016). AM can, in this way, substantially reduce Cd concentrations in plant shoots (Guo et al., 1996; Vivas et al., 2003; Janoušková, et al., 2005; Tan et al., 2015; Zhang et al., 2018), and thereby reduce physical and chemical stress symptoms such as impaired growth, seed production, photosynthesis and antioxidant activity (de Andrade et al., 2008; Tan et al., 2015; Luo et al. 2017). Moreover, mycorrhizal colonisation of the roots may increase with increasing heavy metal content of the soil (Hildebrandt et al., 1999; Audet and Charest, 2006, 4
Hildebrandt et al., 2007). Thus, some plants highly colonised by mycorrhizal fungi have the ability to grow in heavy metal polluted soils (Hildebrandt et al., 1999; Audet and Charest, 2006). Therefore, an intriguing question arises about whether the differences in Cd tolerance and accumulation between plant species depend on the natural variance in the speciesspecific degree of AM-colonisation.
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Previous experiments testing the mycorrhizal effect on plant uptake and tolerance to heavy metals predominantly tested the effects on a single plant species, with or without inoculation with single strains of mycorrhizal fungi, exposed to only two or three metal concentration
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levels (e.g. Joner and Leyval, 1997; Rivera-Becerril et al., 2002; Whitfield et al., 2004; Aloui et al., 2009; Janoušková, et al., 2006). We designed an experiment where six plant species, selected for their natural variation in colonisation, were grown in soil amended with seven
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levels of Cd. Plants were grown under climate-controlled conditions in a greenhouse for 2
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months, measured for chlorophyll content in situ and then destructively harvested for Cd measurements and mycorrhizal colonisation analysis. This design allows us to make a
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complete ecotoxicological assessment of the effect of cadmium on different plant species, and
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accurately test how this is influenced by mycorrhiza. We hypothesized that: (1) Mycorrhizal colonisation will increase with increasing soil Cd concentration to a certain point where the
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conditions for the symbiosis breaks down. (2) Plants colonised with AM will have lower translocation of Cd to shoots than non-mycorrhizal plants due to high root retention. In particular within mycorrhizal plant species there will be a negative correlation between
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colonisation intensity and Cd accumulation in shoots. (3) Mycorrhizal prevention of Cd accumulation in shoots will reduce the ecotoxicological effects of Cd. Thus colonisation will
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enable plants to perform better in Cd polluted soil.
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2. Materials and methods 2.1 Plant species Plant species were selected based on their mycorrhizal status by performing a pilot experiment testing the inoculum potential of the soil and the plants’ degree of mycorrhizal colonisation. We chose six plant species with naturally different colonisation levels that
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covered a gradient in mycorrhizal colonisation: One highly mycorrhizal plant, Hordeum vulgare L. var. Evergreen (>50% colonisation), two with moderate mycorrhizal colonisation, Linum usitatissimum L. (20-50% colonisation) and Sorghum bicolor L. (20-50% colonisation), one with low colonisation, Matricaria recutita L. (<25% colonisation), and two non-mycorrhizal plants, Sinapis alba L. (no colonisation) and Dianthus deltoides L. var. Erectus (no colonisation).
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2.2 Growth medium As foundation for our plant growth medium we used topsoil (0-30 cm) of a sandy clay loam collected from an organic barley field at facilities belonging to the University of Copenhagen
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in Taastrup, Zealand (55.67399°N, 12.28754°E). The soil was passed through a 1-cm metal sieve and mixed thoroughly. Growth medium was then made, by mixing soil with vermiculite and coarse sand 1:1:1 (v/v/v). The resulting growth medium had a pH of 7.2 and a Cd
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background concentration of 0.35 mg kg-1 soil DW. Aliquots of 10 ml CdCl2 (Merc) were mixed
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with the growth medium using a Hercules 6682 mixer (OBH Nordica) to final total concentrations of 0.35, 1.35, 2.85, 6.65, 16.15, 40.15 and 100.35 mg Cd kg-1 soil DW. We chose
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the gradient in accordance with similar studies (Joner and Leyval, 1997; Vivas et al., 2003; Garg
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et al., 2012), and adjusted the levels to fit a logarithmic scale. The Cd-enriched growth medium was stored in plastic bags for a week in a greenhouse at 20 °C to acclimatise before planting.
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Plants were planted in 10x10x10 cm3 pots containing 750 g growth medium. Each plant species and Cd treatment was replicated three times resulting in 6 plant species x 7 Cd levels x 3 replicates = 126 pots in total. As the soil was not sterilised, plants were naturally colonised by
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the indigenous soil mycorrhizal fungi. Since AM fungi have low plant-host specificity (Stukenbrock and Rosendahl, 2005; Santos et al., 2006), we expected the arbuscular
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mycorrhizal fungal community to vary insignificantly between plant species.
2.3 Greenhouse experiment
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Seeds were pre-germinated on moist filter paper. After three days seedlings were selectively picked to obtain uniformity, and planted in pots in numbers according to species-specific size to equalise biomass and intraspecific competition (Hordeum: n=5, Linum: n=9, Sorghum: n=5, Matricaria: n=9, Sinapis: n=9, Dianthus: n=25). The experiment took place in a climatecontrolled greenhouse (20 °C, 16/8 light/dark periods). Pots were randomly placed on 9 plastic trays in clusters of 14 which were interchanged and relocated every 10th day according 6
to a stochastic model. Optimal plant growth was ensured by weekly watering, weeding, and thinning. A standard fertiliser (8.5% K2SO4, 14.7% KHCO3, 18.8% Mg(NO3)2·6H2O, 52.0% Ca(NO3)2·4H2O, 5.9% NH4NO3) was added with the watering. Phosphorus (P) was omitted the first month to allow the mycorrhiza to be fully established. Hereafter, phosphorus (6.2% KH2PO4) was included in the fertiliser to prevent deprivation.
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Plants were destructively harvested after two months. Roots from each pot were thoroughly cleaned with water, cut in 1-cm pieces, randomised, and divided in two subsamples: One for
Cd measurement and one for mycorrhizal colonisation analysis. Some plants growing in the
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most heavily polluted soil were growth-inhibited to such a high degree, that not enough plant
material was available for AM-colonisation analyses (Fig. 1a-f). Immediately before harvesting, the chlorophyll index DUALEX* of Hordeum, Linum, Sorghum, and Sinapis was
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measured with a Dualex Scientific+ polyphenol and chlorophyll-meter (FORCE-A, Orsay,
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France). Chlorophyll index was measured on 3-5 leaves from different plants in each pot and
2.4 Cadmium measurements
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because of their narrow leaves/leaflets.
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averaged. It was not possible to measure the chlorophyll index of Matricaria and Dianthus,
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Roots and shoots were dried at 105 °C for 24 hours. Plant material (0.5 g DW roots and 1.0 g DW shoots) was digested in a MARS 6 Microwave Digestion System (CEM Corporation) with 10 ml 32.5 % nitric acid (HNO3) in Teflon tubes. The samples were then further diluted with
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ddH2O to a final volume of 50 ml and a concentration of 6.5 % nitric acid. Cd concentration of the acid-digested samples was measured using an Atomic Absorption Spectrometer (AAS)
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(PinAAcle™ 900T, Perkin Elmer, USA).
2.5 AMF staining and colonisation
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Root material was cleared with 10% KOH and stained with 0.05% trypan blue according to the modified method of Phillips and Hayman (1970). The stained roots were then stored in lactoglycerol. Colonisation by arbuscular mycorrhizal fungi was estimated as the percentage of root length colonised using the gridline intersect method (McGonigle et al., 1990).
2.6 Data analysis 7
Correlation- and linear regression analyses were performed in SigmaPlot 13.0, and analyses of variance in SAS Enterprise Guide 6.1. To improve linearity, to normalise and to obtain variance homogeneity, some variables were log or arcsine transformed.
3. Results
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3.1 AM-colonisation
The intensity of mycorrhizal colonisation of the six plant species covered a gradient ideal to
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test the effect of colonisation on Cd uptake and translocation. Hordeum was highly colonised (>50%), Linum and Sorghum moderately colonised (25-50%, except at the highest Cd amendment), while Matricaria had a relatively low colonisation (<25%) (Fig. 2). The presumed non-mycorrhizal plants, Sinapis and Dianthus, were only sporadically colonised (0-5 %),
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mostly by septate hyphae, which we classified as non-mycorrhizal endophytic fungi. These
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two plant species differed from the others by their vigorous root hair formation. We saw the
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same Cd-induced pattern in the colonisation of all four mycorrhizal plant species: the colonisation increased with Cd up to a certain level, and then decreased (Fig. 2). The
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colonisation reaches a maximum, which is different for each species. Sorghum and Matricaria were not detectably colonised at the highest Cd levels (Fig. 2). In contrast, Hordeum and
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Linum maintained relatively high colonisation intensity even at the highest Cd amendment.
3.2 The effect of Cd on plant photosynthesis and growth
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Chlorophyll index decreased significantly for Linum (P<0.0001), Hordeum (P=0.0003), and Sinapis (P=0.0093) as a response to increasing Cd levels (Fig. 3). Linum was more photo-
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inhibited by Cd than Hordeum and Sinapis as reflected by the steeper slope of the response curve (Fig. 3). It was not possible to measure the chlorophyll index of Sorghum at the highest
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Cd concentrations because of stunted growth and leaf necrosis. Shoot biomass of all six plant species decreased significantly with increasing Cd levels, but the effect differed markedly between plant species (Fig. 4). Sorghum and Sinapis were most sensitive and had a very similar response to the lowest Cd addition. Matricaria and Linum responded to levels above Cd 6.3 kg-1, while Dianthus and Hordeum still maintained normal
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growth at 15.8 Cd kg-1. Hence, EC50-values varied among species from 8.8 mg Cd kg-1, for the most sensitive species (Sorghum) to 78.6 for Hordeum (Fig. 4).
3.3 Cadmium translocation from roots to shoots Except for two out of 42 treatment combinations (Linum and Sinapis in un-amended soil) shoot concentration of Cd was considerably lower than root concentration (Table 1). Cd
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concentration in shoots of all plant species increased with Cd treatment, however the Cd shoot concentrations showed a trend, where shoots contained un-proportionally more, and more variable amounts of Cd at the highest Cd level (Fig. 5). The highly mycorrhizal Hordeum,
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which showed the highest tolerance to Cd (Fig. 2-4), correspondingly had the lowest concentration of Cd in the shoots at all treatment levels (Fig. 5, Table 1).
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Translocation factor (TF=[Cdshoot]/[Cdroot]); where [Cdshoot] and [Cdroot] is the mean Cd
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concentration (mg Cd kg-1) in the shoots and the roots of a given plant species, is often used as a measure of Cd distribution in plants (Marchiol, 2004; Rezvani and Zaefarian, 2011).
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Despite large variation, a 3-way ANOVA (Table 2) showed that translocation factor depended
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significantly on plant species (P<0.0001), but the degree of mycorrhizal colonisation also had a negative effect on Cd translocation (P=0.02) (Fig. 6). The effect on translocation factor of
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4. Discussion
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soil Cd concentration depended on plant species (P=0.03).
4.1 Experimental approach
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Previous experiments of mycorrhizal effects on plant uptake and tolerance to heavy metals used single plant species, with or without inoculation with particular mycorrhizal strains and
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only few metal concentrations (e.g. Joner and Leyval; 1997; Rivera-Becerril et al. 2002; Whitfield et al. 2004; Aloui et al., 2009; Janoušková, et al., 2006). To improve this generic design we decided not to inoculate plants with single strains of mycorrhizal fungi, but used un-sterilised field soil to create an AM fungal community as natural as possible. This method proved to be ideal for creating natural inoculation, since the four plants expected to be mycorrhizal became well-colonised. The presence of arbuscules in
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the roots demonstrated that symbiotic contact was successfully established for all four plant species. Furthermore, the detailed picture of the combined effect of mycorrhiza, plant species and Cd, confirms the necessity of using an array of Cd concentrations instead of only a few.
4.2 The effect of Cd on AM-colonisation
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According to our first hypothesis we expected Cd to affect mycorrhizal colonisation, in a way where mycorrhizal colonisation would increase with increasing soil Cd concentration until the
conditions for the symbiosis breaks down. Hildebrandt et al. (1999) and Audet and Charest
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(2006) reported mycorrhizal colonisation of roots to increase with increasing heavy metal
content of the soil. Indeed, we also found that a moderate increase in Cd stimulated mycorrhizal development while Cd impeded mycorrhiza at higher concentrations, although,
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more pronounced for some species than others. Our results therefore confirm, that AM is
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facilitated under Cd stress until a certain threshold where the Cd concentration begins to limit the symbiosis. Since the AM symbiont is dependent on plant photosynthates (Chen et al.,
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2005; Cavagnaro, 2008; Lenoir et al., 2016) we suggest, that the mycorrhizal colonisation is
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indirectly affected by high soil concentrations of Cd through the negative effects it has on plant growth. The symbiosis breaks down, when the Cd concentrations in the soil reaches a
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point where the plant can no longer support the mycorrhizal symbiont. This is supported by the EC50-value found for each species, especially for the Cd tolerant Hordeum. Hordeum had the highest EC50-value and was also the plant species most capable of maintaining a high AM-
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colonisation. In contrast, the AM-colonisation of Sorghum decreased already when Cd
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exceeded 16.15 mg/kg soil and had the lowest EC50-value of only 8.5 mg/kg.
4.3 The importance of AM in protecting plants against Cd We found a large difference in Cd uptake between different plant species. Some plants, in
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particular Hordeum, almost completely excluded Cd from their aerial parts, while others translocated extremely high concentrations to the shoots. Cd was generally much higher in the roots than in the shoots indicating that roots are the primary regulation point for plant Cd-translocation. Though, it is not unusual to find Cd-concentrations in roots higher than 10 mg kg-1 DW even at very low soil Cd levels (Hu et al., 2018; Megist et al., 2018), the mycorrhizal Linum had quite high Cd-concentrations in the roots relative to the other plant species in this 10
study. This may be because Linum is a Cd-accumulator species that concentrates Cd in the roots (Schneider and Marquard, 1996; Broadley et al., 2001; Angelova et al. 2004; Jiao et al., 2004). The difference in Cd translocation from roots to shoots depended both on plant species (P=<.0001) and the degree of mycorrhizal colonisation (P=0.0204). Quite interestingly, the most colonised species, Hordeum, also contained the lowest concentration of Cd in the
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shoots.
The correlation between translocation factor and mycorrhizal colonisation exemplifies how
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the mycorrhizal colonisation intensity of each plant species influences the amount of Cd
retained within the root system. We hypothesised that an AM-driven decrease in Cdaccumulation will result in a negative correlation between colonisation intensity and Cd
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accumulation. The decrease in translocation factor with increasing colonisation confirms that
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the differences in Cd-translocation depends on the degree of colonisation. A trend highlighted by the highly mycorrhizal Hordeum which contained 10-20 times less Cd in the shoots than
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any of the other plant species, and correspondingly had the lowest translocation factor at all
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Cd levels. The high Cd-avoidance exhibited by Hordeum seems to be based on strong root retention, keeping Cd within the root system away from the aerial parts of the plant. Even at
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soil Cd-concentration levels of 2.85 mg kg-1, which is not unusual to find in agricultural soils (Nan et al., 2002; Limei et al., 2008; Tóth et al., 2016), Hordeum excludes more than 1000 % of the Cd that enters the roots. Amongst the moderate and low mycorrhizal species, only
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Linum displayed a significant correlation between mycorrhizal colonisation and translocation factor. This may be connected to the observation that this species was most photo-inhibited
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by Cd. Since the photosystems are the primary damage points disrupted by Cd (Küpper et al., 2007; Parmar et al., 2013), Chlorophyll is considered a reliable indicator of Cd toxicity. This
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suggests that Linum is more dependent on mycorrhizal retention than other species.
4.4 Ecotoxicological assessment Our main focus here was the relation between mycorrhizal colonisation, plant uptake, translocation, and response to Cd. We hypothesised that mycorrhizal colonisation prevents Cd-translocation to the shoots and, as a consequence, reduce the ecotoxicological effects caused by Cd, such as impeded growth and chlorosis. Thus AM-colonisation will enable plants 11
to perform better in Cd polluted soil. While we did find that mycorrhizal colonisation affects plant response to Cd, some of the measured indicators of plant vitality such as Chl and EC50, did not correlate as well with the degree of AM colonisation as we expected. Just as it has been reported for many other plant species (Mobin and Khan, 2007; Gill et al., 2012; Chu et al., 2018), our ecotoxicological assessment shows that Cd leads to inhibited photosynthesis and impaired growth. Hence, other factors must be responsible for the differences in Cd-
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tolerance between plant species. We found that the two non-mycorrhizal plants, Sinapis and Dianthus differed from the others by their vigorous root hair formation. Dianthus had a high Cd concentration in the roots but a low shoot concentration. Root hairs increase the
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extension of the roots in the same way as the external hypha of mycorrhizal fungi (Peterson
and Farquhar, 1996; Allen, 2011), thus root hairs might be a factor necessary to consider in
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future studies.
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Despite a high Cd concentration measured in shoots and roots of Sinapis, this species was not strongly affected by high Cd levels. Like other members of the Brassicaceae family, Sinapis is
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known to produce the glucosinolate sinalbin when introduced to elicitors (Pedras and Zaharia,
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2000; Clay et al., 2009; Del Carmen Martinez-Ballesta et al., 2013). It has been found that the concentrations of glucosinolates in the Cd-tolerant hyper-accumulating Thlaspi praecox were
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substantially enhanced compared to the Cd-sensitive species Thlaspi arvense (Tolra et al., 2006), suggesting that glucosinolates may influence Cd tolerance.
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5. Conclusion
Our results support the existence of a protective effect of AM fungi against Cd. A protection
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so efficient, that the concentration of Cd in the shoots is reduced to a much less harmful level. Although not exclusively determining for Cd uptake in plants, we found that Cd translocation between roots and shoots was highly affected by both plant species and the degree of
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mycorrhizal colonisation, especially in plants characterised by very high AM-colonisation. This suggests that protection induced by mycorrhiza, is generally more important than other plant traits, such as vigorous root hair production and protective substances. Thus, the combination of plant species and AM colonisation determine Cd uptake in plants, and should be taken into account when growing plants in Cd-enriched soil.
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Author Contribution Klara Andrés Rask planned and performed the experiment, analysed and interpreted the data and wrote the article, with extensive manual and intellectual input from the other authors in all phases. Flemming Ekelund ([email protected]) takes responsibility for the integrity of
Funding
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the work as a whole, from inception to finished article.
This work was supported by the Danish Council of Strategic Research [DSF-12-132655]; and
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the Danish Council for Independent Research [DFF-4002-00274].
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Declarations of interest: none
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Acknowledgements
This research is part of the ASHBACK project funded by the Danish Council of Strategic
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Research (DSF-12-132655). Klara Andrés Rask, Jesper Liengaard Johansen and Flemming
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Ekelund were funded by Danish Council for Independent Research (DFF-4002-00274).
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Tables:
Table 1: Cadmium (Cd) concentrations in shoots and roots of six plant species grown in soil with seven different levels of Cd1
0.35 mg kg-1
1.35 mg kg-1
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Plant species
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Plant Cd (mg kg-1 DW)
2.85 mg kg-1
Soil Cd (mg kg-1 DW) 6.65 mg kg-1
16.15 mg kg-1
40.15 mg kg-1
100.35 mg kg-1
Shoot Root
0.04 (±0.01) 0.85 (±0.11)
0.69 (±0.34) 13.4 (±0.49)
0.26 (±0.12) 31.5 (±3.10)
6.30 (±0.00) 78.1 (±8.24)
0.64 (±0.12) 155 (±9.83)
6.59 (±0.28) 240 (±16.4)
18.23 (±6.130) 431.5 (±22.81)
Linum usitatissimum
Shoot Root
0.54 (±0.15) 0.41 (±0.11)
7.33 (±0.36) 24.5 (±0.57)
11.0 (±3.03) 57.8 (±4.30)
20.3 (±3.54) 124 (±4.00)
31.7 (±4.43) 210 (±3.98)
39.6 (±1.74) 327 (±46.0)
227.5 (±42.13) 883.0 (±235.8)
Sorghum bicolor
Shoot Root
0.19 (±0.04) 0.68 (±0.08)
7.32 (±1.41) 11.0 (±1.15)
9.49 (±1.44) 19.3 (±0.43)
4.59 (±1.46) 26.0 (±0.70)
8.57 (±2.60) 38.7 (±2.24)
7.37 (±2.11) 45.2 (±4.07)
422.0 (±288.7) 575.1 (±297.7)
Matricaria recutita
Shoot Root
0.25 (±0.05) 0.63 (±0.03)
5.80 (±0.51) 7.74 (±0.52)
5.87 (±1.13) 19.8 (±2.02)
13.0 (±1.44) 83.4 (±8.07)
26.2 (±2.43) 119 (±14.8)
29.3 (±5.21) 124 (±30.2)
208.8 (±32.45) 1040 (±286.0)
Sinapis alba
Shoot Root
0.62 (±0.33) 0.61 (±0.11)
2.78 (±0.18) 13.0 (±0.39)
9.10 (±2.02) 27.7 (±5.31)
11.8 (±3.59) 67.6 (±26.7)
12.8 (±0.49) 151 (±9.81)
23.2 (±2.85) 259 (±6.93)
113.0 (±24.85) 969.7 (±199.3)
Shoot Root
0.08 (±0.02) 0.75 (±0.04)
0.41 (±0.09) 16.4 (±1.95)
1.33 (±0.19) 30.8 (±0.45)
9.06 (±1.99) 84.8 (±4.01)
28.2 (±2.74) 247 (±8.23)
38.3 (±2.68) 421 (±45.9)
162.7 (±39.74) 1214 (±68.10)
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Dianthus deltoides
1Plant
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Hordeum vulgare
material was dried and acid-digested before measurement. Plant Cd concentrations represent the average of three pot replicates.
Standard errors of the mean (±SEM) are shown in brackets. Hordeum vulgare, Linum usitatissimum, Sorghum bicolor, and Matricaria recutita form arbuscular mycorrhiza, whereas Sinapis alba and Dianthus deltoides are non-mycorrhizal.
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Table 2: Summary statistics for the best generalised linear model fitted to the translocation factor (TF = [Cdshoot]/[Cdroot], log transformed)2 denDF
F-value
P-value
Model
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118
18.53
<.0001
AM-colonisation
1
118
5.54
0.0204
Soil Cd concentration
1
118
2.97
0.0876
Plant species
5
118
24.74
Plant species x Soil Cd conc.
5
118
2.56
<.0001
0.0316
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2AM-colonisation
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numDF
(percentage of root length colonised, arcsine transformed), plant species
(classification variable), and soil Cd concentration (mg kg-1, log transformed) were used as
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fixed effects. Significant values (P<0.05) are written in bold.
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Captions: Figure 1: Hordeum vulgare (a), Linum usitatissimum (b), Sorghum bicolor (c), Matricaria recutita (d), Sinapis alba (e), and Dianthus deltoides (f) grown for two months in cadmium
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concentrations of 0.35, 1.35, 2.85, 6.65, 16.15, 40.15 and 100.35 mg Cd kg-1 soil DW.
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Figure 2: Arbuscular mycorrhizal colonisation of four plant species Hordeum vulgare, Linum
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usitatissimum, Sorghum bicolor, and Matricaria recutita, as influenced by cadmium concentration in soil (mg kg-1 ). We expected moderate amounts of cadmium to stimulate
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formation of mycorrhiza and large amounts to impede it. Hence, we used simple 3parameter Gaussian curves to model the results. Values for R2 and P are shown in brackets after the species name. Sinapis alba and Dianthus deltoides also included in the study are
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omitted here, as they did not form mycorrhiza.
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Figure 3: Chlorophyll index (Chl) of Hordeum vulgare, Linum usitatissimum, Sorghum bicolor,
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and Sinapis alba, as influenced by cadmium concentration in soil (mg kg-1). Chlorophyll index was measured in situ directly on the leaves. Linear regression analyses showed that chlorophyll index significantly decreased with soil cadmium concentration (solid lines) for three of the four species. The regression line of Sorghum bicolor is omitted because of
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statistical insignificancy. We were unable to measure the chlorophyll index of Matricaria recutita and Dianthus deltoides due to their small leaves/leaflets. Values for a, R2, and P are
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shown in brackets after the species name.
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Figure 4: Shoot biomass of Hordeum vulgare, Linum usitatissimum, Sorghum bicolor,
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Matricaria recutita, Sinapis alba and Dianthus deltoides as influenced by cadmium concentration in soil (mg kg-1). We considered the relations as doses-response curves of plant tolerance/sensitivity, and therefore used a simple sigmoid function (y = a/(1+exp(x0x)/b)) to explain the relations. Values for R2, P and EC50 (x0) are shown in brackets after the
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species name.
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Figure 5: Cadmium (Cd) concentration in shoots of Hordeum vulgare, Linum usitatissimum,
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Sorghum bicolor, Matricaria recutita, Sinapis alba and Dianthus deltoides as influenced by Cd concentration in soil (mg kg-1). Cd concentration in shoots of all plant species increased with Cd in soil. The Cd shoot concentration showed a trend, where plant Cd-uptake was un-
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proportionally larger at the highest treatment levels. We used a third order equation (P(x)=ax3+bx2+cx+d) for the model, and used the flex point, obtained from the second
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derivative (P´´(x)=0=>x=-b/3a) to indicate at which concentration uptake started to increase
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un-proportionally.
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Figure 6: Translocation factor (TF=[Cdshoot]/[Cdroot]) of cadmium (Cd) from roots to shoots in
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Hordeum vulgare, Linum usitatissimum, Sorghum bicolor, Matricaria recutita, Sinapis alba and Dianthus deltoides shown as correlation between AM-colonisation (arcsine transformed) and TF (log transformed). Lines are only shown where correlations are significant. Solid black line: only mycorrhizal species (a=-1.8, R2=0.60, P<0.0001), dashed
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line: all species (a=-0.86, R2 = 0.24, P<0.0001). Blue line: Linum (a=-1.2, R2=0.26, P=0.023).
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S0098-8472(18)31901-4 https://doi.org/10.1016/j.envexpbot.2019.02.022 EEB 3710
To appear in:
Environmental and Experimental Botany
Received date: Revised date: Accepted date:
21 December 2018 20 February 2019 20 February 2019
Please cite this article as: Rask KA, Johansen JL, Kjøller R, Ekelund F, Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants, Environmental and Experimental Botany (2019), https://doi.org/10.1016/j.envexpbot.2019.02.022 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.
Differences in arbuscular mycorrhizal colonisation influence cadmium uptake in plants
Klara Andrés Rask, Jesper Liengaard Johansen, Rasmus Kjøller, Flemming Ekelund
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Terrestrial Ecology Section, Department of Biology, University of Copenhagen,
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Universitetsparken 15, 2100 Copenhagen Ø, Denmark
Klara Andrés Rask*: [email protected] Jesper Liengaard Johansen: [email protected] Rasmus Kjøller: [email protected]
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Flemming Ekelund: [email protected]
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*Author of correspondence
Date of submission: December 21, 2018
Date of resubmission (major revision): February 13, 2019
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Date of resubmission (minor revision): February 20, 2019
Number of figures: 6
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Image fitting: Figure 1 (2-column), Figure 2 (1-column), Figure 3 (1-column), Figure 4 (1column), Figure 5 (1-column), Figure 6 (1-column)
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Number of tables: 2 Supporting information: 0
Word count: 3341
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Graphical abstract
Highlights
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The combination of plant species and AM colonisation determines Cd uptake in plants. Especially plants with a high degree of colonisation are protected by AM through a high root retention and a low translocation to shoots. Exposure to Cd increases AM colonisation up to a certain threshold where the conditions for the symbiosis breaks down.
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Abstract
Cadmium (Cd) is one of the most toxic heavy metals found in soil. Arbuscular mycorrhiza (AM)
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is known to reduce Cd-translocation in plants by immobilising Cd in the root system. The effect of mycorrhiza on plant Cd-uptake is usually studied in simple systems with single strains
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of mycorrhizal fungi and few levels of Cd. Here we studied how a wide range of soil Cd concentrations affected plant AM-colonisation, and how the species-specific differences in
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AM-colonisation affected uptake, translocation, and toxicity of Cd in plants in a system with naturally occurring mycorrhizal fungi. Six plant species were grown in pots in a greenhouse across seven levels of Cd, which made it possible to model dose-response curves, and calculate EC50 for each plant species. We found a remarkable trend where Cd at moderate levels stimulated mycorrhizal colonisation until a certain threshold where the symbiosis breaks down. Our results support the existence of a protective effect of AM fungi against Cd,
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as the symbiosis reduces Cd-translocation to shoots, especially in plants with very high AMcolonisation. Thus, we conclude that it is the combination of plant species and AM colonisation that determines Cd uptake in plants. AM is therefore an essential trait to consider when growing plants in Cd-polluted soil.
Keywords: Ecotoxicology, heavy metals, mycorrhiza, plant-microbe interactions, bio-
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accumulation, translocation factor
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Abbreviations1
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Arbuscular mycorrhiza (AM), cadmium (Cd)
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1. Introduction Cadmium (Cd) is a heavy metal of major health and environmental concern. It is considered toxic even in small concentrations and has high mobility in food chains (Peralta-Videa et al., 2009; Thévenod and Lee, 2013). Cd pollution mostly originates from the metal industry where it is emitted during the refining process (Halada et al., 2008; Cullen and Maldonado 2013). Cd
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also occurs as a trace element in soil amendments (e.g. phosphate fertilizers, sewage sludge, and wood ash) and may thereby end up in food crops (Jiao et al., 2004; Li et al., 2016). Pollution of agricultural land is not only a problem due to the risk of human and animal consumption
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(Xiong et al., 2014; Zong et al., 2015), but also because of the negative effects Cd has on plant growth even at relatively low soil concentrations (Krämer, 2010; White and Brown, 2010).
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Arbuscular mycorrhizal (AM) fungi form a mutualistic symbiosis with more than 70 % of all
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vascular plant species (Brundrett, 2009; Smith and Read, 2010). The interaction between AM fungi and plants is not a simple relationship, but a continuum of reciprocal benefits depending
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on environmental conditions (Brundrett, 1991; Lekberg et al., 2010; Hempel et al., 2013).
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Plants have intrinsically different degrees of colonisation that vary from consistently high, to intermediate or facultative associations (Zubek and Błaszkowski, 2009; Lekberg et al., 2010;
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Moora, 2014; Brundrett, 2017). Whereas, some plants completely lack the ability to form AM (Koide and Schreiner, 1992; Brundrett, 2004, 2009).
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Mycorrhizal symbiosis increases the uptake of plant nutrients, but may also increase the uptake of non-essential metals (Galli et al., 1994; Leyval et al., 1997; Parmar et al., 2013).
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However, several studies indicate that mycorrhizal colonisation provides plants with an advantage, because toxic elements are immobilised within the mycelia or in the root system (Kaldorf et al., 1999; Joner et al., 2000; Gonzales-Chaves et al., 2004; Hildebrandt et al., 2007;
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Jiang et al., 2016). AM can, in this way, substantially reduce Cd concentrations in plant shoots (Guo et al., 1996; Vivas et al., 2003; Janoušková, et al., 2005; Tan et al., 2015; Zhang et al., 2018), and thereby reduce physical and chemical stress symptoms such as impaired growth, seed production, photosynthesis and antioxidant activity (de Andrade et al., 2008; Tan et al., 2015; Luo et al. 2017). Moreover, mycorrhizal colonisation of the roots may increase with increasing heavy metal content of the soil (Hildebrandt et al., 1999; Audet and Charest, 2006, 4
Hildebrandt et al., 2007). Thus, some plants highly colonised by mycorrhizal fungi have the ability to grow in heavy metal polluted soils (Hildebrandt et al., 1999; Audet and Charest, 2006). Therefore, an intriguing question arises about whether the differences in Cd tolerance and accumulation between plant species depend on the natural variance in the speciesspecific degree of AM-colonisation.
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Previous experiments testing the mycorrhizal effect on plant uptake and tolerance to heavy metals predominantly tested the effects on a single plant species, with or without inoculation with single strains of mycorrhizal fungi, exposed to only two or three metal concentration
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levels (e.g. Joner and Leyval, 1997; Rivera-Becerril et al., 2002; Whitfield et al., 2004; Aloui et al., 2009; Janoušková, et al., 2006). We designed an experiment where six plant species, selected for their natural variation in colonisation, were grown in soil amended with seven
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levels of Cd. Plants were grown under climate-controlled conditions in a greenhouse for 2
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months, measured for chlorophyll content in situ and then destructively harvested for Cd measurements and mycorrhizal colonisation analysis. This design allows us to make a
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complete ecotoxicological assessment of the effect of cadmium on different plant species, and
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accurately test how this is influenced by mycorrhiza. We hypothesized that: (1) Mycorrhizal colonisation will increase with increasing soil Cd concentration to a certain point where the
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conditions for the symbiosis breaks down. (2) Plants colonised with AM will have lower translocation of Cd to shoots than non-mycorrhizal plants due to high root retention. In particular within mycorrhizal plant species there will be a negative correlation between
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colonisation intensity and Cd accumulation in shoots. (3) Mycorrhizal prevention of Cd accumulation in shoots will reduce the ecotoxicological effects of Cd. Thus colonisation will
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enable plants to perform better in Cd polluted soil.
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2. Materials and methods 2.1 Plant species Plant species were selected based on their mycorrhizal status by performing a pilot experiment testing the inoculum potential of the soil and the plants’ degree of mycorrhizal colonisation. We chose six plant species with naturally different colonisation levels that
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covered a gradient in mycorrhizal colonisation: One highly mycorrhizal plant, Hordeum vulgare L. var. Evergreen (>50% colonisation), two with moderate mycorrhizal colonisation, Linum usitatissimum L. (20-50% colonisation) and Sorghum bicolor L. (20-50% colonisation), one with low colonisation, Matricaria recutita L. (<25% colonisation), and two non-mycorrhizal plants, Sinapis alba L. (no colonisation) and Dianthus deltoides L. var. Erectus (no colonisation).
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2.2 Growth medium As foundation for our plant growth medium we used topsoil (0-30 cm) of a sandy clay loam collected from an organic barley field at facilities belonging to the University of Copenhagen
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in Taastrup, Zealand (55.67399°N, 12.28754°E). The soil was passed through a 1-cm metal sieve and mixed thoroughly. Growth medium was then made, by mixing soil with vermiculite and coarse sand 1:1:1 (v/v/v). The resulting growth medium had a pH of 7.2 and a Cd
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background concentration of 0.35 mg kg-1 soil DW. Aliquots of 10 ml CdCl2 (Merc) were mixed
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with the growth medium using a Hercules 6682 mixer (OBH Nordica) to final total concentrations of 0.35, 1.35, 2.85, 6.65, 16.15, 40.15 and 100.35 mg Cd kg-1 soil DW. We chose
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the gradient in accordance with similar studies (Joner and Leyval, 1997; Vivas et al., 2003; Garg
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et al., 2012), and adjusted the levels to fit a logarithmic scale. The Cd-enriched growth medium was stored in plastic bags for a week in a greenhouse at 20 °C to acclimatise before planting.
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Plants were planted in 10x10x10 cm3 pots containing 750 g growth medium. Each plant species and Cd treatment was replicated three times resulting in 6 plant species x 7 Cd levels x 3 replicates = 126 pots in total. As the soil was not sterilised, plants were naturally colonised by
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the indigenous soil mycorrhizal fungi. Since AM fungi have low plant-host specificity (Stukenbrock and Rosendahl, 2005; Santos et al., 2006), we expected the arbuscular
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mycorrhizal fungal community to vary insignificantly between plant species.
2.3 Greenhouse experiment
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Seeds were pre-germinated on moist filter paper. After three days seedlings were selectively picked to obtain uniformity, and planted in pots in numbers according to species-specific size to equalise biomass and intraspecific competition (Hordeum: n=5, Linum: n=9, Sorghum: n=5, Matricaria: n=9, Sinapis: n=9, Dianthus: n=25). The experiment took place in a climatecontrolled greenhouse (20 °C, 16/8 light/dark periods). Pots were randomly placed on 9 plastic trays in clusters of 14 which were interchanged and relocated every 10th day according 6
to a stochastic model. Optimal plant growth was ensured by weekly watering, weeding, and thinning. A standard fertiliser (8.5% K2SO4, 14.7% KHCO3, 18.8% Mg(NO3)2·6H2O, 52.0% Ca(NO3)2·4H2O, 5.9% NH4NO3) was added with the watering. Phosphorus (P) was omitted the first month to allow the mycorrhiza to be fully established. Hereafter, phosphorus (6.2% KH2PO4) was included in the fertiliser to prevent deprivation.
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Plants were destructively harvested after two months. Roots from each pot were thoroughly cleaned with water, cut in 1-cm pieces, randomised, and divided in two subsamples: One for
Cd measurement and one for mycorrhizal colonisation analysis. Some plants growing in the
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most heavily polluted soil were growth-inhibited to such a high degree, that not enough plant
material was available for AM-colonisation analyses (Fig. 1a-f). Immediately before harvesting, the chlorophyll index DUALEX* of Hordeum, Linum, Sorghum, and Sinapis was
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measured with a Dualex Scientific+ polyphenol and chlorophyll-meter (FORCE-A, Orsay,
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France). Chlorophyll index was measured on 3-5 leaves from different plants in each pot and
2.4 Cadmium measurements
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because of their narrow leaves/leaflets.
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averaged. It was not possible to measure the chlorophyll index of Matricaria and Dianthus,
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Roots and shoots were dried at 105 °C for 24 hours. Plant material (0.5 g DW roots and 1.0 g DW shoots) was digested in a MARS 6 Microwave Digestion System (CEM Corporation) with 10 ml 32.5 % nitric acid (HNO3) in Teflon tubes. The samples were then further diluted with
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ddH2O to a final volume of 50 ml and a concentration of 6.5 % nitric acid. Cd concentration of the acid-digested samples was measured using an Atomic Absorption Spectrometer (AAS)
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(PinAAcle™ 900T, Perkin Elmer, USA).
2.5 AMF staining and colonisation
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Root material was cleared with 10% KOH and stained with 0.05% trypan blue according to the modified method of Phillips and Hayman (1970). The stained roots were then stored in lactoglycerol. Colonisation by arbuscular mycorrhizal fungi was estimated as the percentage of root length colonised using the gridline intersect method (McGonigle et al., 1990).
2.6 Data analysis 7
Correlation- and linear regression analyses were performed in SigmaPlot 13.0, and analyses of variance in SAS Enterprise Guide 6.1. To improve linearity, to normalise and to obtain variance homogeneity, some variables were log or arcsine transformed.
3. Results
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3.1 AM-colonisation
The intensity of mycorrhizal colonisation of the six plant species covered a gradient ideal to
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test the effect of colonisation on Cd uptake and translocation. Hordeum was highly colonised (>50%), Linum and Sorghum moderately colonised (25-50%, except at the highest Cd amendment), while Matricaria had a relatively low colonisation (<25%) (Fig. 2). The presumed non-mycorrhizal plants, Sinapis and Dianthus, were only sporadically colonised (0-5 %),
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mostly by septate hyphae, which we classified as non-mycorrhizal endophytic fungi. These
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two plant species differed from the others by their vigorous root hair formation. We saw the
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same Cd-induced pattern in the colonisation of all four mycorrhizal plant species: the colonisation increased with Cd up to a certain level, and then decreased (Fig. 2). The
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colonisation reaches a maximum, which is different for each species. Sorghum and Matricaria were not detectably colonised at the highest Cd levels (Fig. 2). In contrast, Hordeum and
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Linum maintained relatively high colonisation intensity even at the highest Cd amendment.
3.2 The effect of Cd on plant photosynthesis and growth
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Chlorophyll index decreased significantly for Linum (P<0.0001), Hordeum (P=0.0003), and Sinapis (P=0.0093) as a response to increasing Cd levels (Fig. 3). Linum was more photo-
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inhibited by Cd than Hordeum and Sinapis as reflected by the steeper slope of the response curve (Fig. 3). It was not possible to measure the chlorophyll index of Sorghum at the highest
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Cd concentrations because of stunted growth and leaf necrosis. Shoot biomass of all six plant species decreased significantly with increasing Cd levels, but the effect differed markedly between plant species (Fig. 4). Sorghum and Sinapis were most sensitive and had a very similar response to the lowest Cd addition. Matricaria and Linum responded to levels above Cd 6.3 kg-1, while Dianthus and Hordeum still maintained normal
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growth at 15.8 Cd kg-1. Hence, EC50-values varied among species from 8.8 mg Cd kg-1, for the most sensitive species (Sorghum) to 78.6 for Hordeum (Fig. 4).
3.3 Cadmium translocation from roots to shoots Except for two out of 42 treatment combinations (Linum and Sinapis in un-amended soil) shoot concentration of Cd was considerably lower than root concentration (Table 1). Cd
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concentration in shoots of all plant species increased with Cd treatment, however the Cd shoot concentrations showed a trend, where shoots contained un-proportionally more, and more variable amounts of Cd at the highest Cd level (Fig. 5). The highly mycorrhizal Hordeum,
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which showed the highest tolerance to Cd (Fig. 2-4), correspondingly had the lowest concentration of Cd in the shoots at all treatment levels (Fig. 5, Table 1).
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Translocation factor (TF=[Cdshoot]/[Cdroot]); where [Cdshoot] and [Cdroot] is the mean Cd
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concentration (mg Cd kg-1) in the shoots and the roots of a given plant species, is often used as a measure of Cd distribution in plants (Marchiol, 2004; Rezvani and Zaefarian, 2011).
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Despite large variation, a 3-way ANOVA (Table 2) showed that translocation factor depended
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significantly on plant species (P<0.0001), but the degree of mycorrhizal colonisation also had a negative effect on Cd translocation (P=0.02) (Fig. 6). The effect on translocation factor of
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4. Discussion
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soil Cd concentration depended on plant species (P=0.03).
4.1 Experimental approach
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Previous experiments of mycorrhizal effects on plant uptake and tolerance to heavy metals used single plant species, with or without inoculation with particular mycorrhizal strains and
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only few metal concentrations (e.g. Joner and Leyval; 1997; Rivera-Becerril et al. 2002; Whitfield et al. 2004; Aloui et al., 2009; Janoušková, et al., 2006). To improve this generic design we decided not to inoculate plants with single strains of mycorrhizal fungi, but used un-sterilised field soil to create an AM fungal community as natural as possible. This method proved to be ideal for creating natural inoculation, since the four plants expected to be mycorrhizal became well-colonised. The presence of arbuscules in
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the roots demonstrated that symbiotic contact was successfully established for all four plant species. Furthermore, the detailed picture of the combined effect of mycorrhiza, plant species and Cd, confirms the necessity of using an array of Cd concentrations instead of only a few.
4.2 The effect of Cd on AM-colonisation
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According to our first hypothesis we expected Cd to affect mycorrhizal colonisation, in a way where mycorrhizal colonisation would increase with increasing soil Cd concentration until the
conditions for the symbiosis breaks down. Hildebrandt et al. (1999) and Audet and Charest
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(2006) reported mycorrhizal colonisation of roots to increase with increasing heavy metal
content of the soil. Indeed, we also found that a moderate increase in Cd stimulated mycorrhizal development while Cd impeded mycorrhiza at higher concentrations, although,
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more pronounced for some species than others. Our results therefore confirm, that AM is
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facilitated under Cd stress until a certain threshold where the Cd concentration begins to limit the symbiosis. Since the AM symbiont is dependent on plant photosynthates (Chen et al.,
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2005; Cavagnaro, 2008; Lenoir et al., 2016) we suggest, that the mycorrhizal colonisation is
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indirectly affected by high soil concentrations of Cd through the negative effects it has on plant growth. The symbiosis breaks down, when the Cd concentrations in the soil reaches a
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point where the plant can no longer support the mycorrhizal symbiont. This is supported by the EC50-value found for each species, especially for the Cd tolerant Hordeum. Hordeum had the highest EC50-value and was also the plant species most capable of maintaining a high AM-
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colonisation. In contrast, the AM-colonisation of Sorghum decreased already when Cd
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exceeded 16.15 mg/kg soil and had the lowest EC50-value of only 8.5 mg/kg.
4.3 The importance of AM in protecting plants against Cd We found a large difference in Cd uptake between different plant species. Some plants, in
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particular Hordeum, almost completely excluded Cd from their aerial parts, while others translocated extremely high concentrations to the shoots. Cd was generally much higher in the roots than in the shoots indicating that roots are the primary regulation point for plant Cd-translocation. Though, it is not unusual to find Cd-concentrations in roots higher than 10 mg kg-1 DW even at very low soil Cd levels (Hu et al., 2018; Megist et al., 2018), the mycorrhizal Linum had quite high Cd-concentrations in the roots relative to the other plant species in this 10
study. This may be because Linum is a Cd-accumulator species that concentrates Cd in the roots (Schneider and Marquard, 1996; Broadley et al., 2001; Angelova et al. 2004; Jiao et al., 2004). The difference in Cd translocation from roots to shoots depended both on plant species (P=<.0001) and the degree of mycorrhizal colonisation (P=0.0204). Quite interestingly, the most colonised species, Hordeum, also contained the lowest concentration of Cd in the
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shoots.
The correlation between translocation factor and mycorrhizal colonisation exemplifies how
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the mycorrhizal colonisation intensity of each plant species influences the amount of Cd
retained within the root system. We hypothesised that an AM-driven decrease in Cdaccumulation will result in a negative correlation between colonisation intensity and Cd
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accumulation. The decrease in translocation factor with increasing colonisation confirms that
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the differences in Cd-translocation depends on the degree of colonisation. A trend highlighted by the highly mycorrhizal Hordeum which contained 10-20 times less Cd in the shoots than
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any of the other plant species, and correspondingly had the lowest translocation factor at all
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Cd levels. The high Cd-avoidance exhibited by Hordeum seems to be based on strong root retention, keeping Cd within the root system away from the aerial parts of the plant. Even at
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soil Cd-concentration levels of 2.85 mg kg-1, which is not unusual to find in agricultural soils (Nan et al., 2002; Limei et al., 2008; Tóth et al., 2016), Hordeum excludes more than 1000 % of the Cd that enters the roots. Amongst the moderate and low mycorrhizal species, only
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Linum displayed a significant correlation between mycorrhizal colonisation and translocation factor. This may be connected to the observation that this species was most photo-inhibited
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by Cd. Since the photosystems are the primary damage points disrupted by Cd (Küpper et al., 2007; Parmar et al., 2013), Chlorophyll is considered a reliable indicator of Cd toxicity. This
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suggests that Linum is more dependent on mycorrhizal retention than other species.
4.4 Ecotoxicological assessment Our main focus here was the relation between mycorrhizal colonisation, plant uptake, translocation, and response to Cd. We hypothesised that mycorrhizal colonisation prevents Cd-translocation to the shoots and, as a consequence, reduce the ecotoxicological effects caused by Cd, such as impeded growth and chlorosis. Thus AM-colonisation will enable plants 11
to perform better in Cd polluted soil. While we did find that mycorrhizal colonisation affects plant response to Cd, some of the measured indicators of plant vitality such as Chl and EC50, did not correlate as well with the degree of AM colonisation as we expected. Just as it has been reported for many other plant species (Mobin and Khan, 2007; Gill et al., 2012; Chu et al., 2018), our ecotoxicological assessment shows that Cd leads to inhibited photosynthesis and impaired growth. Hence, other factors must be responsible for the differences in Cd-
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tolerance between plant species. We found that the two non-mycorrhizal plants, Sinapis and Dianthus differed from the others by their vigorous root hair formation. Dianthus had a high Cd concentration in the roots but a low shoot concentration. Root hairs increase the
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extension of the roots in the same way as the external hypha of mycorrhizal fungi (Peterson
and Farquhar, 1996; Allen, 2011), thus root hairs might be a factor necessary to consider in
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future studies.
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Despite a high Cd concentration measured in shoots and roots of Sinapis, this species was not strongly affected by high Cd levels. Like other members of the Brassicaceae family, Sinapis is
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known to produce the glucosinolate sinalbin when introduced to elicitors (Pedras and Zaharia,
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2000; Clay et al., 2009; Del Carmen Martinez-Ballesta et al., 2013). It has been found that the concentrations of glucosinolates in the Cd-tolerant hyper-accumulating Thlaspi praecox were
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substantially enhanced compared to the Cd-sensitive species Thlaspi arvense (Tolra et al., 2006), suggesting that glucosinolates may influence Cd tolerance.
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5. Conclusion
Our results support the existence of a protective effect of AM fungi against Cd. A protection
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so efficient, that the concentration of Cd in the shoots is reduced to a much less harmful level. Although not exclusively determining for Cd uptake in plants, we found that Cd translocation between roots and shoots was highly affected by both plant species and the degree of
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mycorrhizal colonisation, especially in plants characterised by very high AM-colonisation. This suggests that protection induced by mycorrhiza, is generally more important than other plant traits, such as vigorous root hair production and protective substances. Thus, the combination of plant species and AM colonisation determine Cd uptake in plants, and should be taken into account when growing plants in Cd-enriched soil.
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Author Contribution Klara Andrés Rask planned and performed the experiment, analysed and interpreted the data and wrote the article, with extensive manual and intellectual input from the other authors in all phases. Flemming Ekelund ([email protected]) takes responsibility for the integrity of
Funding
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the work as a whole, from inception to finished article.
This work was supported by the Danish Council of Strategic Research [DSF-12-132655]; and
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the Danish Council for Independent Research [DFF-4002-00274].
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Declarations of interest: none
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Acknowledgements
This research is part of the ASHBACK project funded by the Danish Council of Strategic
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Research (DSF-12-132655). Klara Andrés Rask, Jesper Liengaard Johansen and Flemming
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Ekelund were funded by Danish Council for Independent Research (DFF-4002-00274).
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Tables:
Table 1: Cadmium (Cd) concentrations in shoots and roots of six plant species grown in soil with seven different levels of Cd1
0.35 mg kg-1
1.35 mg kg-1
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Plant species
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Plant Cd (mg kg-1 DW)
2.85 mg kg-1
Soil Cd (mg kg-1 DW) 6.65 mg kg-1
16.15 mg kg-1
40.15 mg kg-1
100.35 mg kg-1
Shoot Root
0.04 (±0.01) 0.85 (±0.11)
0.69 (±0.34) 13.4 (±0.49)
0.26 (±0.12) 31.5 (±3.10)
6.30 (±0.00) 78.1 (±8.24)
0.64 (±0.12) 155 (±9.83)
6.59 (±0.28) 240 (±16.4)
18.23 (±6.130) 431.5 (±22.81)
Linum usitatissimum
Shoot Root
0.54 (±0.15) 0.41 (±0.11)
7.33 (±0.36) 24.5 (±0.57)
11.0 (±3.03) 57.8 (±4.30)
20.3 (±3.54) 124 (±4.00)
31.7 (±4.43) 210 (±3.98)
39.6 (±1.74) 327 (±46.0)
227.5 (±42.13) 883.0 (±235.8)
Sorghum bicolor
Shoot Root
0.19 (±0.04) 0.68 (±0.08)
7.32 (±1.41) 11.0 (±1.15)
9.49 (±1.44) 19.3 (±0.43)
4.59 (±1.46) 26.0 (±0.70)
8.57 (±2.60) 38.7 (±2.24)
7.37 (±2.11) 45.2 (±4.07)
422.0 (±288.7) 575.1 (±297.7)
Matricaria recutita
Shoot Root
0.25 (±0.05) 0.63 (±0.03)
5.80 (±0.51) 7.74 (±0.52)
5.87 (±1.13) 19.8 (±2.02)
13.0 (±1.44) 83.4 (±8.07)
26.2 (±2.43) 119 (±14.8)
29.3 (±5.21) 124 (±30.2)
208.8 (±32.45) 1040 (±286.0)
Sinapis alba
Shoot Root
0.62 (±0.33) 0.61 (±0.11)
2.78 (±0.18) 13.0 (±0.39)
9.10 (±2.02) 27.7 (±5.31)
11.8 (±3.59) 67.6 (±26.7)
12.8 (±0.49) 151 (±9.81)
23.2 (±2.85) 259 (±6.93)
113.0 (±24.85) 969.7 (±199.3)
Shoot Root
0.08 (±0.02) 0.75 (±0.04)
0.41 (±0.09) 16.4 (±1.95)
1.33 (±0.19) 30.8 (±0.45)
9.06 (±1.99) 84.8 (±4.01)
28.2 (±2.74) 247 (±8.23)
38.3 (±2.68) 421 (±45.9)
162.7 (±39.74) 1214 (±68.10)
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Dianthus deltoides
1Plant
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Hordeum vulgare
material was dried and acid-digested before measurement. Plant Cd concentrations represent the average of three pot replicates.
Standard errors of the mean (±SEM) are shown in brackets. Hordeum vulgare, Linum usitatissimum, Sorghum bicolor, and Matricaria recutita form arbuscular mycorrhiza, whereas Sinapis alba and Dianthus deltoides are non-mycorrhizal.
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Table 2: Summary statistics for the best generalised linear model fitted to the translocation factor (TF = [Cdshoot]/[Cdroot], log transformed)2 denDF
F-value
P-value
Model
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118
18.53
<.0001
AM-colonisation
1
118
5.54
0.0204
Soil Cd concentration
1
118
2.97
0.0876
Plant species
5
118
24.74
Plant species x Soil Cd conc.
5
118
2.56
<.0001
0.0316
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2AM-colonisation
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numDF
(percentage of root length colonised, arcsine transformed), plant species
(classification variable), and soil Cd concentration (mg kg-1, log transformed) were used as
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fixed effects. Significant values (P<0.05) are written in bold.
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Captions: Figure 1: Hordeum vulgare (a), Linum usitatissimum (b), Sorghum bicolor (c), Matricaria recutita (d), Sinapis alba (e), and Dianthus deltoides (f) grown for two months in cadmium
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concentrations of 0.35, 1.35, 2.85, 6.65, 16.15, 40.15 and 100.35 mg Cd kg-1 soil DW.
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Figure 2: Arbuscular mycorrhizal colonisation of four plant species Hordeum vulgare, Linum
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usitatissimum, Sorghum bicolor, and Matricaria recutita, as influenced by cadmium concentration in soil (mg kg-1 ). We expected moderate amounts of cadmium to stimulate
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formation of mycorrhiza and large amounts to impede it. Hence, we used simple 3parameter Gaussian curves to model the results. Values for R2 and P are shown in brackets after the species name. Sinapis alba and Dianthus deltoides also included in the study are
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omitted here, as they did not form mycorrhiza.
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Figure 3: Chlorophyll index (Chl) of Hordeum vulgare, Linum usitatissimum, Sorghum bicolor,
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and Sinapis alba, as influenced by cadmium concentration in soil (mg kg-1). Chlorophyll index was measured in situ directly on the leaves. Linear regression analyses showed that chlorophyll index significantly decreased with soil cadmium concentration (solid lines) for three of the four species. The regression line of Sorghum bicolor is omitted because of
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statistical insignificancy. We were unable to measure the chlorophyll index of Matricaria recutita and Dianthus deltoides due to their small leaves/leaflets. Values for a, R2, and P are
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shown in brackets after the species name.
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Figure 4: Shoot biomass of Hordeum vulgare, Linum usitatissimum, Sorghum bicolor,
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Matricaria recutita, Sinapis alba and Dianthus deltoides as influenced by cadmium concentration in soil (mg kg-1). We considered the relations as doses-response curves of plant tolerance/sensitivity, and therefore used a simple sigmoid function (y = a/(1+exp(x0x)/b)) to explain the relations. Values for R2, P and EC50 (x0) are shown in brackets after the
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species name.
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Figure 5: Cadmium (Cd) concentration in shoots of Hordeum vulgare, Linum usitatissimum,
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Sorghum bicolor, Matricaria recutita, Sinapis alba and Dianthus deltoides as influenced by Cd concentration in soil (mg kg-1). Cd concentration in shoots of all plant species increased with Cd in soil. The Cd shoot concentration showed a trend, where plant Cd-uptake was un-
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proportionally larger at the highest treatment levels. We used a third order equation (P(x)=ax3+bx2+cx+d) for the model, and used the flex point, obtained from the second
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derivative (P´´(x)=0=>x=-b/3a) to indicate at which concentration uptake started to increase
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un-proportionally.
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Figure 6: Translocation factor (TF=[Cdshoot]/[Cdroot]) of cadmium (Cd) from roots to shoots in
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Hordeum vulgare, Linum usitatissimum, Sorghum bicolor, Matricaria recutita, Sinapis alba and Dianthus deltoides shown as correlation between AM-colonisation (arcsine transformed) and TF (log transformed). Lines are only shown where correlations are significant. Solid black line: only mycorrhizal species (a=-1.8, R2=0.60, P<0.0001), dashed
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line: all species (a=-0.86, R2 = 0.24, P<0.0001). Blue line: Linum (a=-1.2, R2=0.26, P=0.023).
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