Chemosphere 124 (2015) 110–115
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Morphological and physiological responses of maize (Zea mays) exposed to sand contaminated by phenanthrene Joan Dupuy, Stéphanie Ouvrard, Pierre Leglize ⇑, Thibault Sterckeman Université de Lorraine, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy F-54518, France INRA, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy F-54518, France
h i g h l i g h t s 1
From 50 mg kg
, phenanthrene induces perturbations in maize functioning.
It causes a modification of the carbon allocation, favouring the roots. It modifies the root architecture, making the roots thicker on average. Symptoms suggest water shortage, photosynthesis slowdown, nutritional perturbation.
a r t i c l e
i n f o
Article history: Received 28 February 2014 Received in revised form 16 October 2014 Accepted 18 November 2014 Available online 12 December 2014 Handling Editor: A. Gies Keywords: Phytotoxicity PAH Mineral nutrition Root morphology
a b s t r a c t Phytoremediation is promising, but depends on clearly understanding contaminants’ impact on plant functioning. We therefore focused on the impact of polycyclic aromatic hydrocarbons (PAH) on cultivated plants and understanding the impact of phenanthrene (PHE) on maize functioning (Zea mays). Cultivation was conducted under controlled conditions on artificially contaminated sand with PHE levels increasing from 50 to 750 mg PHE kg1. After four weeks, plants exposed to levels above 50 mg PHE kg1 presented decreased biomasses and reduced photosynthetic activity. These modifications were associated with higher biomass allocations to roots and lower ones to stems. The leaf biomass proportion was similar, with thinner blades than controls. PHE-exposed plant showed modified root architecture, with fewer roots of 0.2 and 0.4 mm in diameter. Leaves were potassium-deplete, but calcium, phosphorus, magnesium and zinc-enriched. Their content in nitrogen, iron, sulfur and manganese was unaffected. These responses resembled those of water-stress, although water contents in plant organs were not affected by PHE and water supply was not limited. They also indicated a possible perturbation of both nutritional functioning and photosynthesis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Of the wide diversity of metallic and organic pollutants, polycyclic aromatic hydrocarbons (PAH) are among the most worrying because of their ubiquity and toxicity. They are persistent organic pollutants (POP), conferring them the ability to accumulate into organisms and to resist in-soil degradation. Nowadays, PAH-contaminated soil remediation technologies include solvent extraction, chemical oxidation and thermal treatment (Gan et al., 2009). In phytoremediation, a less conventional option, the upper plant is
⇑ Corresponding author at: Laboratoire Sols et Environnement, Université de Lorraine/INRA, 2 avenue de la Forêt de Haye, TSA 40602, F-54518 Vandœuvrelès-Nancy cedex, France. Tel.: +33 383 595 761; fax: +33 383 595 791. E-mail address: [email protected] (P. Leglize). http://dx.doi.org/10.1016/j.chemosphere.2014.11.051 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
used to contain, degrade or remove pollutants (Sterckeman et al., 2012), offering a cost-effective and environmentally-friendly in-situ cleanup technology (Ouvrard et al., 2014). Rhizodegradation, the principal phytoremediation process, consists in an enhanced microbial degradation activity in the rhizosphere zone. However, the establishment of vegetation cover on these contaminated soils may provide additional benefits such as landscaping or valuable biomass production (Licht and Isebrands, 2005). The fate of PAH in soils is well-documented (Pignatello and Xing, 1996; Alexander, 2000; Semple et al., 2003; Ouvrard et al., 2014). Efficiency of plant-assisted degradation for PAH has been proved with controlled laboratory conditions (Chang and Corapcioglu, 1998; Binet et al., 2000; Joner and Leyval, 2003). However, this bioremediation technology has proved hard to apply at the field scale (Ouvrard et al., 2011) due to difficulties in growing and developing
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plants. Many symptoms have been reported, including germination inhibition and decreased biomass production (Henner et al., 1999; Kummerová and Kmentová, 2004; Smith et al., 2006; Kummerová ˇ ová et al., 2012), reduced photosynthetic pigment content (Ván et al., 2009; Oguntimehin et al., 2010), formation of reactive oxygen species (Alkio et al., 2005; Pašková et al., 2006; Liu et al., 2009), thicker and shorter roots (Kummerová and Kmentová, 2004; Merkl et al., 2005; Kummerová et al., 2013). Yet the intoxication mechanism of PAH in plants remains mostly unknown. Insight into pollutant-impact on plants is essential for vegetating PAHcontaminated sites and may help improve agronomic practices and plant species choice for improving productivity and pollutant degradation. The aim of this study was to assess morphological and physiological changes of (Zea mays L.) exposed to sand spiked with phenanthrene (PHE). Maize and phenanthrene were selected as models, as this plant species has a fast and biomass-productive growth, a large fasciculate root system and has already been studied during PAH exposure (Lin et al., 2007; Kummerová et al., 2012, 2013). Besides, maize is widely cropped in agriculture and is of economic interest. PHE is among the 16 EPA priority PAH and is commonly used in laboratory essays (Pašková et al., 2006; Gao and Collins, 2009; Liu et al., 2009; Zhan et al., 2010; Desalme et al., 2011). Cultivation was conducted for four weeks in a growth chamber under controlled conditions on artificially contaminated sand with levels of PHE increasing from 50 to 750 mg PHE kg1. Maize growth and organ development were assessed, by measuring both its morphological parameters i.e. biomass, biomass allocation, root architecture and physiological functions i.e. photosynthesis and transpiration by measuring CO2 and H2O fluxes and nutrition by quantifying mineral nutrient uptake.
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2.3. Experimental set-up Cultivation was conducted in glass jars, each containing 2 kg of dry sand. They were wrapped in dark plastic sheeting to protect substrate and roots from light exposure. One seedling was transplanted into each glass jar with 0, 50, 150, 250, 500 or 750 mg PHE kg1 in six replicates. Jars without seedlings contained sand contaminated at 0, 150 and 750 mg PHE kg1 with three replicates. All jars were fertilized at 80% water-holding capacity (WHC) with adapted Ruakura nutrient solution (Smith et al., 1983), at a pH value of 6.5. The cultivation lasted 28 d, in the following conditions: 16 h of light (325 lmol photons m2 s1), 20/18 °C day/night temperatures and 70% relative air humidity. Water content was maintained by weighing and adding deionized water every two days and nutrient solution every three additions. 2.4. Measurement of morphological parameters At the end of the cultivation, the fresh stem, root, and leaf biomass was weighed separately (FBM, in g). Roots were scanned and their architecture analyzed using the WinRhizoÒ software (Regent Instruments Inc., Québec, Qc, Canada). Visually, necrotic part of each leaf was separated from non-necrotic part. Necrotic and non-necrotic areas were measured with an electronic leaf-area meter (LI-3000, Li-Cor Inc., Lincoln, Nebraska, USA). Sum of the two was referred to as leaf area (LA, in m2). All the plant samples were freeze-dried for dry biomass measurement (DBM, in g) of each organ. Relative water content (RWC, %) of each plant was determined according to:
RWC ¼
FBM DBM 100 FBM
ð1Þ
Biomass allocation (BApo, %) to a plant organ was assessed as: 2. Materials and methods
BApo ¼ 2.1. Growth medium Cultivation was conducted in sand (Ø 0.4–0.8 mm, Sibelco, Hostun, France), which was spiked with phenanthrene to achieve six different contamination levels: 50, 150, 250, 500 and 750 mg kg1 dw. Spiked sand was chosen as growth medium to maximize PHE availability. PHE stock solution (>97%, Acros Organics) was prepared at 43.1 g L1 in HPLC grade dichloromethane. To achieve homogenous contamination, for each contamination level, a sub-sample of dry sand, representing 10% of the required total mass was spiked at 16% v/w by a PHE solution diluted from the stock solution so as to achieve the final desired concentrations of 50, 150, 250, 500 and 750 mg kg1. The spiked sand was placed under a laboratory fume hood until complete solvent evaporation, and homogenized with the remaining fraction of non-contaminated sand by quartering. The control treatment was spiked with the same volume of pure dichloromethane and treated similarly.
DBMpo 100; DBMT
ð2Þ
where DBMpo was the plant organ dry biomass and DBMT was the total plant dry biomass. Morphological parameters were measured, i.e. specific leaf area (SLA, m2 kg1).
SLA ¼
LA 103 ; leaf DBM
ð3Þ
where Leaf DBM was the leaf dry biomass (g). The estimated leaf thickness (ELT, lm) was calculated (Vile et al., 2005):
ELT ¼
1 SLA LDMC
ð4Þ
where LDMC is the leaf dry matter content (leaf dry biomass/leaf fresh biomass mg g1). 2.5. Leaf gas exchanges
2.2. Plant material Before use, maize seeds (Zea mays L., cv MB862, INRA, Saint Martin de Hinx, France) were sterilized with TFD9 detergent (Didecyldimethylammonium chlorid, tetrapotassium Ethylenediamine-tetra-acetate, C11-15 secondary alcohol ethoxyles, n-(3aminopropyl)-n-dodecylpropane-1,3-diamine, potassium hydroxide – Franklab, France) (v/v 20%) for 15 min and rinsed once with deionized water. They were then exposed to hydrogen peroxide (v/v 10%) for 3 min and rinsed three times with deionized water. They were pre-germinated on watered cotton in a dark room for two days at room temperature before experimentation.
Photosynthesis and transpiration were determined by measuring CO2 (lmol m2 s1) and H2O (mmol m2 s1) fluxes with a portable gas exchange system (Li-Cor 6200, Li-Cor Inc., Lincoln, Nebraska, USA) on the second leaf. Measurements were performed one time after 25 d of cultivation, when leafs were enough developed. 2.6. Mineral nutrient uptake Micro and macro element contents were measured in roots and leaves. Dry matter was milled at 200 lm in an agate mortar. A 0.1 g test portion of dry biomass was first digested in a glass tube with
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2 mL HNO3 (5 M) for 12 h at ambient temperature and 24 h at 80 °C. After cooling, 2 mL of H2O2 (3 M) were added and residual organic matter was digested again for 48 h at ambient temperature. The extracts were filtered on ashless paper filters and adjusted to 10 mL. Micro (Fe, Mn and Zn) and macronutrient (K, Ca, Mg, P and S) contents were measured using Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES, Thermo Scientific, USA). Control materials, from Noccaea caerulescens and maize, with known compositions (internal analyses carried out by INRA-USRAVE, Villenave d’Ornon, France) were included in analyses for quantitative verification of the results. Nitrogen content was determined through dry combustion (Vario Micro Cube, Elementar, France). 2.7. Statistical analysis R Software (version 3.0.1, R Development Core Team, 2011) was used for data processing. After verification of normality with Shapiro-Wilk’s test, comparison between treatments for each parameters were made using a one-way ANOVA followed by Tukey’s HSD test or Kruskal–Wallis test were respectively used for parametric or non-parametric distributions to compare differences between multiple groups. 3. Results 3.1. Organ morphology Among the 6 replicates for each treatment, for plants exposed to 50, 150, 250, 500 and 750 mg PHE kg1 sand (dw) the number of
living plants after 28 d of growth was respectively 4, 4, 4, 4, 5 and 6 for control treatment. Despite the high survival rate, maize plants exposed to PHE showed a much lower DBMT than the control plants, representing up to 7% of the control DBMT (Table 1). The DBMT production was affected at the first PHE exposure level (50 mg PHE kg1) being 75% lower than the control. In these higher exposure, ELT was 39–49% lower, while it was only 17.8% lower at 50 mg kg1. Plant RWC did not present any difference and was around 90.5% for all treatments. Leaf morphology, SLA, which was around 43 m2 kg1 in the control treatment, was not significantly different for plants exposed to PHE. Leaves from plants exposed to PHE also presented increasingly necrotic areas on their tips, from 14% to 31% of total surface. In contrast, ELT was significantly higher in control plants than in those exposed to PHE concentrations exceeding 50 mg kg1. In these treatments, ELT was only 18% lower at 50 mg kg1, whilst for treatments above 150 mg kg1, it was 39–49% lower. Differences in dry mass allocation to plant parts were also observed. The proportion of roots rose significantly to around 29% of the total dry biomass for plants exposed to PHE, while this was 20% for control plants. However, this was independent of the increasing PHE exposure level. PHE-exposure also caused a significant decrease in stem proportion, falling from 27% in control plants, and decreasing from 21% to 15% with levels of PHE increasing from 50 to 750 mg PHE kg1. The leaf proportion remained approximately 54% of the total dry biomass whatever the exposure level. Root morphology, displayed a significant tendency to increase in diameter with PHE exposure levels (Fig. 1). Most control plant root diameters were smaller than 0.2 mm (52% of the total length),
Table 1 Mean values ± standard errors of total dry biomass (DBMT), relative water content (RWC), specific leaf area (SLA), necrosed area of leaves and estimated leaf thickness (ELT) of maize (Zea mays) after a 28 d cultivation in sand and nutrient solution with different initial phenanthrene concentrations. Gas exchanges of CO2 and H2O at the second leaf of maize (Zea mays) after a 25 d cultivation. n is the number of replicates. Mean values with the same letter are not significantly different (p < 0.05). [PHE]initial (mg kg1 dw)
n
DBMT (g)
RWC (% mass)
0 50 150 250 500 750
6 4 4 4 4 5
0.88 ± 0 .14 a 0.22 ± 0.05 b 0.08 ± 0.01 b 0.07 ± 0.02 b 0.11 ± 0.02 b 0.06 ± 0.01 b
89.9 ± 1.1 92.4 ± 1.3 89.4 ± 0.8 90.4 ± 1.2 89.7 ± 0.8 91.2 ± 0.0
SLA (m2 kg1) a a a a a a
43.1 ± 4.0 53.5 ± 1.3 54.2 ± 3.4 51.4 ± 5.5 49.5 ± 5.5 53.7 ± 4.8
a a a a a a
Necrotic area (% LA)
ELT (lm)
CO2 input (lmol m2 s1)
H2O output (mmol m2 s1)
0±0 b 1.6 ± 1.0 ab 31 ± 12.7 a 18 ± 3.4 ab 14 ± 2.1 ab 19 ± 4.5 a
166 ± 3 a 136 ± 7 ab 101 ± 12 bc 85 ± 13 c 102 ± 9 bc 96 ± 6 c
0.88 ± 0.10 0.43 ± 0.15 0.14 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.04 ± 0.02
0.17 ± 0.03 0.16 ± 0.03 0.05 ± 0.05 0.05 ± 0.02 0.08 ± 0.02 0.11 ± 0.02
a ab bc c c c
a a a a a a
Fig. 1. Total root length distribution of maize (Zea mays) according to the root diameter (%) after a 28 d cultivation in sand and nutrient solution with different initial phenanthrene concentrations. Error bars stand for standard errors, n = 6. For each class of diameter, mean values with the same letter are not significantly different (p < 0.05).
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although 26% had diameters between 0.2 and 0.4 mm. However, these proportions appeared to reverse with increasing PHE levels. More plants had the thinnest roots (diameter < 0.2 mm) at the lowest two PHE concentrations, representing 41% and 43% of the total root length for 50 and 150 mg kg1 PHE, whilst from 250 mg kg1 PHE and above, roots of the thinnest diameter were significantly fewer, accounting for 23–27% of the total length. The proportion of the thickest roots, with diameters between 1.2 and 1.8 mm, also decreased from 2.7% in control plants to 0.4% at 750 mg kg1 PHE. In contrast, the proportion of roots with a 0.2–0.4 mm diameter significantly increased to 39% at 250 mg kg1 PHE, while that of roots of the three diameter classes between 0.4 and 1.2 mm all slightly increased. 3.2. Gas exchange Maize leaves exposed to PHE showed a significant decrease in CO2 absorption, reaching 51% at 50 mg PHE kg1 (0.43 lmol m2 s1), 84% at 150 mg kg1 (0.14 lmol m2 s1) and 98% for PHE levels between 250 and 750 mg kg1 (around 0.03 lmol m2 s1), less than controls (0.88 lmol m2 s1). The same trend was observed for H2O emissions, although differences were not statistically significant. 3.3. Mineral nutrition PHE-exposed roots showed no great difference in nutrient contents, despite significant decreases in N and K at 750 mg kg1, of respectively 60% and 56%. Roots exposed to between 150 and 500 mg kg1 presented lower Ca and Mn contents than both controls and plants exposed to 750 mg kg1 PHE (Table 2). Plants leaves exposed to levels above 50 mg PHE kg1 tended to accumulate higher concentrations of Ca, Mg, P and Zn than control plants, respectively +48%, +32%, +68%, and +49% (Table 2). PHE-exposed plant leaves also had significantly depleted K content, i.e. around 77%, less the than control value. Other leaf nutrient contents including N, S, Fe and Mn were unaffected by PHE exposure. 4. Discussion At the end of the experiment, PHE concentration in sand was very low (below 50 lg kg1, data not shown). We assumed that maize response was induced by the early effects of exposure to PHE or its metabolites. Plant responses to PHE were not proportional to PHE concentrations. Indeed, similar responses were observed between 150 and 750 mg PHE kg1, while they were less pronounced for the 50 mg kg1 PHE exposure. This suggests that PAH exposure did not induce a general shut down of maize
development at low concentrations. This trend was in agreement with that observed by Alkio et al. (2005), who reported increasing stress symptoms at low PHE concentrations (between 0.05 and 0.25 mM) and equal responses at greater exposure (from 0.25 to 0.75 mM) of Arabidopsis thaliana grown on a Petri dish containing solid growth medium. PHE exposure inhibited maize growth even at low concentrations (50 mg kg1) (Table 1), which is in agreement with results obtained with other plant species such as A. thaliana (Alkio et al., 2005), rice (Li et al., 2008) and tomato (Ahammed et al., 2012). However, our results provide new insights into biomass allocation as a response to PHE exposure. Indeed, in this study PHE-exposed maize seemed to preserve both foliar and root system allocation, yet producing thinner leaves, at the expense of the stem. These morphological modifications would indicate that maize compensates for PHE exposure to optimize photosynthesis: With greater leaf surface per unit biomass, plants would apparently maximize light exposure and gas-exchange for photosynthetic cells. However, measurements showed decreased transpiration and CO2 absorption in plants exposed to PHE concentrations exceeding 50 mg kg1. This reduced photosynthetic carbon fixation could be the reason for lower biomass production. Concerning the root system, root biomass allocation was significantly higher for maize exposed to PHE and was accompanied by a thickening of root system, which is in agreement with the findings of Kummerová et al. (2013). These morphological changes to the root system were observed for all PHE exposure levels. Root nutrient content was unaffected by PHE exposure, except for the highest exposure level which showed N and K depletions, and S, Mn and Fe accumulations. Preservation of leaf biomass in the case of PHE exposure was accompanied by a modification to the nutrient composition with K+ depletion, Ca2+, Mg2+, P and Zn2+ accumulations, yet no significant modifications to the contents of the other major elements. Potassium and calcium are known to be involved in the stomata’s functioning. Indeed potassium ion-release from guard cells is triggered by elevation of the intracellular Ca2+ concentration, and causes stomatal pore closing (Ward and Schroeder, 1994) as in the case of water-stress plant response. Furthermore, some symptoms (decreased biomass and CO2 input, high Mg2+ concentrations related to reduced photosynthesis) reported in the present study are found in plants subjected to water-stress (Rao et al., 1987; Tezara et al., 1999; Lawlor, 2002; Shao et al., 2008). However in this study, sand moisture was the same for each treatment (80% water-holding capacity) and relative water contents in roots, stems and leaves of PHE-exposed plants showed no difference to that of the control treatment. A water-stress type response has already appeared as a symptom of PAH and/or fuel oil hydrocarbon exposure (Adam and
Table 2 Mean values ± standard errors of nutrient concentrations (lmol g1 dw) in roots of maize (Zea mays) after a 28 d cultivation in sand and nutrient solution with different phenanthrene concentrations. n is the number of replicates. For each element and each plant organ mean values with the same letter are not significantly different (p < 0.05). [PHE] initial (mg kg1 dw)
n
N
Leaves
0 50 150 250 500 750
6 3 4 4 4 4
3283 ± 269 4070 ± 242 3598 ± 125 2737 ± 728 2958 ± 564 2954 ± 636
Roots
0 50 150 250 500 750
6 3 3 3 4 3
2189 ± 142 2573 ± 150 2190 ± 202 2797 ± 164 2348 ± 149 957 ± 120
n
K
Ca
a a a a a a
6 4 4 4 4 5
413 ± 62 a 567 ± 87 a 188 ± 47 b 100 ± 10 b 87 ± 9 b 81 ± 8 b
152 ± 11 250 ± 28 243 ± 26 306 ± 36 285 ± 20 328 ± 12
b a ab a a a
a a a a a b
6 3 4 4 4 5
230 ± 25 253 ± 72 194 ± 30 123 ± 16 136 ± 19 106 ± 21
209 ± 11 161 ± 18 122 ± 21 133 ± 18 110 ± 19 200 ± 20
a abc c bc c ab
a a ab ab ab b
Mg
P
83 ± 4 c 97 ± 6 bc 96 ± 11 bc 108 ± 10 ab 118 ± 6 ab 142 ± 9 a
70 ± 10 129 ± 21 152 ± 25 189 ± 20 183 ± 29 277 ± 25
59 ± 3 40 ± 4 37 ± 9 37 ± 7 25 ± 5 43 ± 7
a ab ab ab b ab
S c bc bc ab ab a
73 ± 5 a 86 ± 9 a 85 ± 3 a 74 ± 9 a 63 ± 3 a 84 ± 13 a
90 ± 5 b 119 ± 10 a 89 ± 9 b 91 ± 10 b 83 ± 6 ab 103 ± 7 b 82 ± 2 ab 85 ± 1 ab 91 ± 16 ab 72 ± 4 b 72 ± 5 b 103 ± 4 a
Fe 2.3 ± 0.2 3.1 ± 0.5 3.6 ± 0.4 5.0 ± 1.4 2.9 ± 0.2 3.1 ± 0.2
Mn a a a a a a
4.2 ± 0.4 a 6.8 ± 0.4 ab 10.1 ± 2.2 b 6.2 ± 0.4 ab 8.4 ± 0.5 b 24.5 ± 14.4 b
0.57 ± 0.07 0.90 ± 0.08 0.71 ± 0.05 0.79 ± 0.04 0.70 ± 0.02 0.91 ± 0.11
Zn b a ab ab ab a
0.81 ± 0.11 1.14 ± 0.06 1.72 ± 0.13 1.55 ± 0.21 1.39 ± 0.09 1.72 ± 0.06
c bc a ab ab a
0.41 ± 0.06 ab 0.32 ± 0.02 ab 0.28 ± 0.0 b 0.26 ± 0.03 b 0.27 ± 0.04 b 0.60 ± 0.10 a
1.57 ± 0.32 0.97 ± 0.10 1.63 ± 0.30 1.38 ± 0.05 1.23 ± 0.21 1.43 ± 0.19
a a a a a a
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Duncan, 2002; Merkl et al., 2005; Langer et al., 2010). A plant’s defense mechanisms to water-stress can also disturb oxidant defense (Jiang and Zhang, 2002). This may be related to oxidative stress coupled with PAH exposure as reported in several studies (Alkio et al., 2005; Pašková et al., 2006; Liu et al., 2009), resulting in tissue and cell damage. Hypothetically, the PHE molecule or its downstream products may induce phytohormone abscisic acid ˇ ová et al., 2009), or interact with (ABA) molecule production (Ván ABA receptors in Zea mays, leading to water-stress responses. ABA plays a major role in this chemical signaling (Pospíšilová, 2003). Weisman et al. (2010) revealed slight correlations with PHE and ABA gene expression in A. thaliana, but showed an involvement of phyto-hormones and defense signaling pathways in response to PAHs. Consequently, PHE or its metabolites might have impacted the plant’s physiology on various levels, including its nutritional processes, as revealed by the concentration modifications to some nutrients in both leaves and roots (Osakabe et al., 2014). 5. Conclusion Phenanthrene appeared to be highly toxic to maize. Plant growth was altered when the substrate contained 50 mg kg1. However, plants showed an initial adaptive response to PHE toxicity, through a greater allocation of carbon assimilates to roots, a similar one to leaves and a lower one to stems. PHE-exposure caused the roots to be thicker and the leaves thinner. Maize plants exposed to PHE presented symptoms of the water-stress type response although the plants suffered no water supply limitation. PHE exposure modified mineral plant nutrition with increasing calcium content in leaves. Calcium is known to be involved in plant signaling and environmental stress response in terms of growth and development. These results provide a new perspective for plant growth on contaminated soil in a phytoremediation context, especially for the agronomic management of a phytoremediation process. The development of the root system seemed crucial and further investigation is needed to assess the effect of PHE exposure to the roots’ inner structure (e.g. apoplastic barriers, xylem or phloem vessels). Acknowledgement This work was financially supported by the Région Lorraine and SNOWMAN IBRACS project. The authors thank Christophe Robin for his advice and for the Li-Cor6200 machine, Romain Goudon for his help in ICP analysis, as well as Lucas Charrois and Rémi Baldos for plant biomass conditioning. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.11.051. References Adam, G., Duncan, H., 2002. Influence of diesel fuel on seed germination. Environ. Pollut. 120, 363–370. Ahammed, G.J., Yuan, H.-L., Ogweno, J.O., Zhou, Y.-H., Xia, X.-J., Mao, W.-H., Shi, K., Yu, J.-Q., 2012. Brassinosteroid alleviates phenanthrene and pyrene phytotoxicity by increasing detoxification activity and photosynthesis in tomato. Chemosphere 86, 546–555. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol. 34, 4259–4265. Alkio, M., Tabuchi, T.M., Wang, X., Colón-Carmona, A., 2005. Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms. J. Exp. Bot. 56, 2983–2994.
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Morphological and physiological responses of maize (Zea mays) exposed to sand contaminated by phenanthrene Joan Dupuy, Stéphanie Ouvrard, Pierre Leglize ⇑, Thibault Sterckeman Université de Lorraine, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy F-54518, France INRA, Laboratoire Sols et Environnement, UMR 1120, Vandœuvre-lès-Nancy F-54518, France
h i g h l i g h t s 1
From 50 mg kg
, phenanthrene induces perturbations in maize functioning.
It causes a modification of the carbon allocation, favouring the roots. It modifies the root architecture, making the roots thicker on average. Symptoms suggest water shortage, photosynthesis slowdown, nutritional perturbation.
a r t i c l e
i n f o
Article history: Received 28 February 2014 Received in revised form 16 October 2014 Accepted 18 November 2014 Available online 12 December 2014 Handling Editor: A. Gies Keywords: Phytotoxicity PAH Mineral nutrition Root morphology
a b s t r a c t Phytoremediation is promising, but depends on clearly understanding contaminants’ impact on plant functioning. We therefore focused on the impact of polycyclic aromatic hydrocarbons (PAH) on cultivated plants and understanding the impact of phenanthrene (PHE) on maize functioning (Zea mays). Cultivation was conducted under controlled conditions on artificially contaminated sand with PHE levels increasing from 50 to 750 mg PHE kg1. After four weeks, plants exposed to levels above 50 mg PHE kg1 presented decreased biomasses and reduced photosynthetic activity. These modifications were associated with higher biomass allocations to roots and lower ones to stems. The leaf biomass proportion was similar, with thinner blades than controls. PHE-exposed plant showed modified root architecture, with fewer roots of 0.2 and 0.4 mm in diameter. Leaves were potassium-deplete, but calcium, phosphorus, magnesium and zinc-enriched. Their content in nitrogen, iron, sulfur and manganese was unaffected. These responses resembled those of water-stress, although water contents in plant organs were not affected by PHE and water supply was not limited. They also indicated a possible perturbation of both nutritional functioning and photosynthesis. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Of the wide diversity of metallic and organic pollutants, polycyclic aromatic hydrocarbons (PAH) are among the most worrying because of their ubiquity and toxicity. They are persistent organic pollutants (POP), conferring them the ability to accumulate into organisms and to resist in-soil degradation. Nowadays, PAH-contaminated soil remediation technologies include solvent extraction, chemical oxidation and thermal treatment (Gan et al., 2009). In phytoremediation, a less conventional option, the upper plant is
⇑ Corresponding author at: Laboratoire Sols et Environnement, Université de Lorraine/INRA, 2 avenue de la Forêt de Haye, TSA 40602, F-54518 Vandœuvrelès-Nancy cedex, France. Tel.: +33 383 595 761; fax: +33 383 595 791. E-mail address: [email protected] (P. Leglize). http://dx.doi.org/10.1016/j.chemosphere.2014.11.051 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
used to contain, degrade or remove pollutants (Sterckeman et al., 2012), offering a cost-effective and environmentally-friendly in-situ cleanup technology (Ouvrard et al., 2014). Rhizodegradation, the principal phytoremediation process, consists in an enhanced microbial degradation activity in the rhizosphere zone. However, the establishment of vegetation cover on these contaminated soils may provide additional benefits such as landscaping or valuable biomass production (Licht and Isebrands, 2005). The fate of PAH in soils is well-documented (Pignatello and Xing, 1996; Alexander, 2000; Semple et al., 2003; Ouvrard et al., 2014). Efficiency of plant-assisted degradation for PAH has been proved with controlled laboratory conditions (Chang and Corapcioglu, 1998; Binet et al., 2000; Joner and Leyval, 2003). However, this bioremediation technology has proved hard to apply at the field scale (Ouvrard et al., 2011) due to difficulties in growing and developing
J. Dupuy et al. / Chemosphere 124 (2015) 110–115
plants. Many symptoms have been reported, including germination inhibition and decreased biomass production (Henner et al., 1999; Kummerová and Kmentová, 2004; Smith et al., 2006; Kummerová ˇ ová et al., 2012), reduced photosynthetic pigment content (Ván et al., 2009; Oguntimehin et al., 2010), formation of reactive oxygen species (Alkio et al., 2005; Pašková et al., 2006; Liu et al., 2009), thicker and shorter roots (Kummerová and Kmentová, 2004; Merkl et al., 2005; Kummerová et al., 2013). Yet the intoxication mechanism of PAH in plants remains mostly unknown. Insight into pollutant-impact on plants is essential for vegetating PAHcontaminated sites and may help improve agronomic practices and plant species choice for improving productivity and pollutant degradation. The aim of this study was to assess morphological and physiological changes of (Zea mays L.) exposed to sand spiked with phenanthrene (PHE). Maize and phenanthrene were selected as models, as this plant species has a fast and biomass-productive growth, a large fasciculate root system and has already been studied during PAH exposure (Lin et al., 2007; Kummerová et al., 2012, 2013). Besides, maize is widely cropped in agriculture and is of economic interest. PHE is among the 16 EPA priority PAH and is commonly used in laboratory essays (Pašková et al., 2006; Gao and Collins, 2009; Liu et al., 2009; Zhan et al., 2010; Desalme et al., 2011). Cultivation was conducted for four weeks in a growth chamber under controlled conditions on artificially contaminated sand with levels of PHE increasing from 50 to 750 mg PHE kg1. Maize growth and organ development were assessed, by measuring both its morphological parameters i.e. biomass, biomass allocation, root architecture and physiological functions i.e. photosynthesis and transpiration by measuring CO2 and H2O fluxes and nutrition by quantifying mineral nutrient uptake.
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2.3. Experimental set-up Cultivation was conducted in glass jars, each containing 2 kg of dry sand. They were wrapped in dark plastic sheeting to protect substrate and roots from light exposure. One seedling was transplanted into each glass jar with 0, 50, 150, 250, 500 or 750 mg PHE kg1 in six replicates. Jars without seedlings contained sand contaminated at 0, 150 and 750 mg PHE kg1 with three replicates. All jars were fertilized at 80% water-holding capacity (WHC) with adapted Ruakura nutrient solution (Smith et al., 1983), at a pH value of 6.5. The cultivation lasted 28 d, in the following conditions: 16 h of light (325 lmol photons m2 s1), 20/18 °C day/night temperatures and 70% relative air humidity. Water content was maintained by weighing and adding deionized water every two days and nutrient solution every three additions. 2.4. Measurement of morphological parameters At the end of the cultivation, the fresh stem, root, and leaf biomass was weighed separately (FBM, in g). Roots were scanned and their architecture analyzed using the WinRhizoÒ software (Regent Instruments Inc., Québec, Qc, Canada). Visually, necrotic part of each leaf was separated from non-necrotic part. Necrotic and non-necrotic areas were measured with an electronic leaf-area meter (LI-3000, Li-Cor Inc., Lincoln, Nebraska, USA). Sum of the two was referred to as leaf area (LA, in m2). All the plant samples were freeze-dried for dry biomass measurement (DBM, in g) of each organ. Relative water content (RWC, %) of each plant was determined according to:
RWC ¼
FBM DBM 100 FBM
ð1Þ
Biomass allocation (BApo, %) to a plant organ was assessed as: 2. Materials and methods
BApo ¼ 2.1. Growth medium Cultivation was conducted in sand (Ø 0.4–0.8 mm, Sibelco, Hostun, France), which was spiked with phenanthrene to achieve six different contamination levels: 50, 150, 250, 500 and 750 mg kg1 dw. Spiked sand was chosen as growth medium to maximize PHE availability. PHE stock solution (>97%, Acros Organics) was prepared at 43.1 g L1 in HPLC grade dichloromethane. To achieve homogenous contamination, for each contamination level, a sub-sample of dry sand, representing 10% of the required total mass was spiked at 16% v/w by a PHE solution diluted from the stock solution so as to achieve the final desired concentrations of 50, 150, 250, 500 and 750 mg kg1. The spiked sand was placed under a laboratory fume hood until complete solvent evaporation, and homogenized with the remaining fraction of non-contaminated sand by quartering. The control treatment was spiked with the same volume of pure dichloromethane and treated similarly.
DBMpo 100; DBMT
ð2Þ
where DBMpo was the plant organ dry biomass and DBMT was the total plant dry biomass. Morphological parameters were measured, i.e. specific leaf area (SLA, m2 kg1).
SLA ¼
LA 103 ; leaf DBM
ð3Þ
where Leaf DBM was the leaf dry biomass (g). The estimated leaf thickness (ELT, lm) was calculated (Vile et al., 2005):
ELT ¼
1 SLA LDMC
ð4Þ
where LDMC is the leaf dry matter content (leaf dry biomass/leaf fresh biomass mg g1). 2.5. Leaf gas exchanges
2.2. Plant material Before use, maize seeds (Zea mays L., cv MB862, INRA, Saint Martin de Hinx, France) were sterilized with TFD9 detergent (Didecyldimethylammonium chlorid, tetrapotassium Ethylenediamine-tetra-acetate, C11-15 secondary alcohol ethoxyles, n-(3aminopropyl)-n-dodecylpropane-1,3-diamine, potassium hydroxide – Franklab, France) (v/v 20%) for 15 min and rinsed once with deionized water. They were then exposed to hydrogen peroxide (v/v 10%) for 3 min and rinsed three times with deionized water. They were pre-germinated on watered cotton in a dark room for two days at room temperature before experimentation.
Photosynthesis and transpiration were determined by measuring CO2 (lmol m2 s1) and H2O (mmol m2 s1) fluxes with a portable gas exchange system (Li-Cor 6200, Li-Cor Inc., Lincoln, Nebraska, USA) on the second leaf. Measurements were performed one time after 25 d of cultivation, when leafs were enough developed. 2.6. Mineral nutrient uptake Micro and macro element contents were measured in roots and leaves. Dry matter was milled at 200 lm in an agate mortar. A 0.1 g test portion of dry biomass was first digested in a glass tube with
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2 mL HNO3 (5 M) for 12 h at ambient temperature and 24 h at 80 °C. After cooling, 2 mL of H2O2 (3 M) were added and residual organic matter was digested again for 48 h at ambient temperature. The extracts were filtered on ashless paper filters and adjusted to 10 mL. Micro (Fe, Mn and Zn) and macronutrient (K, Ca, Mg, P and S) contents were measured using Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES, Thermo Scientific, USA). Control materials, from Noccaea caerulescens and maize, with known compositions (internal analyses carried out by INRA-USRAVE, Villenave d’Ornon, France) were included in analyses for quantitative verification of the results. Nitrogen content was determined through dry combustion (Vario Micro Cube, Elementar, France). 2.7. Statistical analysis R Software (version 3.0.1, R Development Core Team, 2011) was used for data processing. After verification of normality with Shapiro-Wilk’s test, comparison between treatments for each parameters were made using a one-way ANOVA followed by Tukey’s HSD test or Kruskal–Wallis test were respectively used for parametric or non-parametric distributions to compare differences between multiple groups. 3. Results 3.1. Organ morphology Among the 6 replicates for each treatment, for plants exposed to 50, 150, 250, 500 and 750 mg PHE kg1 sand (dw) the number of
living plants after 28 d of growth was respectively 4, 4, 4, 4, 5 and 6 for control treatment. Despite the high survival rate, maize plants exposed to PHE showed a much lower DBMT than the control plants, representing up to 7% of the control DBMT (Table 1). The DBMT production was affected at the first PHE exposure level (50 mg PHE kg1) being 75% lower than the control. In these higher exposure, ELT was 39–49% lower, while it was only 17.8% lower at 50 mg kg1. Plant RWC did not present any difference and was around 90.5% for all treatments. Leaf morphology, SLA, which was around 43 m2 kg1 in the control treatment, was not significantly different for plants exposed to PHE. Leaves from plants exposed to PHE also presented increasingly necrotic areas on their tips, from 14% to 31% of total surface. In contrast, ELT was significantly higher in control plants than in those exposed to PHE concentrations exceeding 50 mg kg1. In these treatments, ELT was only 18% lower at 50 mg kg1, whilst for treatments above 150 mg kg1, it was 39–49% lower. Differences in dry mass allocation to plant parts were also observed. The proportion of roots rose significantly to around 29% of the total dry biomass for plants exposed to PHE, while this was 20% for control plants. However, this was independent of the increasing PHE exposure level. PHE-exposure also caused a significant decrease in stem proportion, falling from 27% in control plants, and decreasing from 21% to 15% with levels of PHE increasing from 50 to 750 mg PHE kg1. The leaf proportion remained approximately 54% of the total dry biomass whatever the exposure level. Root morphology, displayed a significant tendency to increase in diameter with PHE exposure levels (Fig. 1). Most control plant root diameters were smaller than 0.2 mm (52% of the total length),
Table 1 Mean values ± standard errors of total dry biomass (DBMT), relative water content (RWC), specific leaf area (SLA), necrosed area of leaves and estimated leaf thickness (ELT) of maize (Zea mays) after a 28 d cultivation in sand and nutrient solution with different initial phenanthrene concentrations. Gas exchanges of CO2 and H2O at the second leaf of maize (Zea mays) after a 25 d cultivation. n is the number of replicates. Mean values with the same letter are not significantly different (p < 0.05). [PHE]initial (mg kg1 dw)
n
DBMT (g)
RWC (% mass)
0 50 150 250 500 750
6 4 4 4 4 5
0.88 ± 0 .14 a 0.22 ± 0.05 b 0.08 ± 0.01 b 0.07 ± 0.02 b 0.11 ± 0.02 b 0.06 ± 0.01 b
89.9 ± 1.1 92.4 ± 1.3 89.4 ± 0.8 90.4 ± 1.2 89.7 ± 0.8 91.2 ± 0.0
SLA (m2 kg1) a a a a a a
43.1 ± 4.0 53.5 ± 1.3 54.2 ± 3.4 51.4 ± 5.5 49.5 ± 5.5 53.7 ± 4.8
a a a a a a
Necrotic area (% LA)
ELT (lm)
CO2 input (lmol m2 s1)
H2O output (mmol m2 s1)
0±0 b 1.6 ± 1.0 ab 31 ± 12.7 a 18 ± 3.4 ab 14 ± 2.1 ab 19 ± 4.5 a
166 ± 3 a 136 ± 7 ab 101 ± 12 bc 85 ± 13 c 102 ± 9 bc 96 ± 6 c
0.88 ± 0.10 0.43 ± 0.15 0.14 ± 0.01 0.01 ± 0.01 0.03 ± 0.01 0.04 ± 0.02
0.17 ± 0.03 0.16 ± 0.03 0.05 ± 0.05 0.05 ± 0.02 0.08 ± 0.02 0.11 ± 0.02
a ab bc c c c
a a a a a a
Fig. 1. Total root length distribution of maize (Zea mays) according to the root diameter (%) after a 28 d cultivation in sand and nutrient solution with different initial phenanthrene concentrations. Error bars stand for standard errors, n = 6. For each class of diameter, mean values with the same letter are not significantly different (p < 0.05).
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although 26% had diameters between 0.2 and 0.4 mm. However, these proportions appeared to reverse with increasing PHE levels. More plants had the thinnest roots (diameter < 0.2 mm) at the lowest two PHE concentrations, representing 41% and 43% of the total root length for 50 and 150 mg kg1 PHE, whilst from 250 mg kg1 PHE and above, roots of the thinnest diameter were significantly fewer, accounting for 23–27% of the total length. The proportion of the thickest roots, with diameters between 1.2 and 1.8 mm, also decreased from 2.7% in control plants to 0.4% at 750 mg kg1 PHE. In contrast, the proportion of roots with a 0.2–0.4 mm diameter significantly increased to 39% at 250 mg kg1 PHE, while that of roots of the three diameter classes between 0.4 and 1.2 mm all slightly increased. 3.2. Gas exchange Maize leaves exposed to PHE showed a significant decrease in CO2 absorption, reaching 51% at 50 mg PHE kg1 (0.43 lmol m2 s1), 84% at 150 mg kg1 (0.14 lmol m2 s1) and 98% for PHE levels between 250 and 750 mg kg1 (around 0.03 lmol m2 s1), less than controls (0.88 lmol m2 s1). The same trend was observed for H2O emissions, although differences were not statistically significant. 3.3. Mineral nutrition PHE-exposed roots showed no great difference in nutrient contents, despite significant decreases in N and K at 750 mg kg1, of respectively 60% and 56%. Roots exposed to between 150 and 500 mg kg1 presented lower Ca and Mn contents than both controls and plants exposed to 750 mg kg1 PHE (Table 2). Plants leaves exposed to levels above 50 mg PHE kg1 tended to accumulate higher concentrations of Ca, Mg, P and Zn than control plants, respectively +48%, +32%, +68%, and +49% (Table 2). PHE-exposed plant leaves also had significantly depleted K content, i.e. around 77%, less the than control value. Other leaf nutrient contents including N, S, Fe and Mn were unaffected by PHE exposure. 4. Discussion At the end of the experiment, PHE concentration in sand was very low (below 50 lg kg1, data not shown). We assumed that maize response was induced by the early effects of exposure to PHE or its metabolites. Plant responses to PHE were not proportional to PHE concentrations. Indeed, similar responses were observed between 150 and 750 mg PHE kg1, while they were less pronounced for the 50 mg kg1 PHE exposure. This suggests that PAH exposure did not induce a general shut down of maize
development at low concentrations. This trend was in agreement with that observed by Alkio et al. (2005), who reported increasing stress symptoms at low PHE concentrations (between 0.05 and 0.25 mM) and equal responses at greater exposure (from 0.25 to 0.75 mM) of Arabidopsis thaliana grown on a Petri dish containing solid growth medium. PHE exposure inhibited maize growth even at low concentrations (50 mg kg1) (Table 1), which is in agreement with results obtained with other plant species such as A. thaliana (Alkio et al., 2005), rice (Li et al., 2008) and tomato (Ahammed et al., 2012). However, our results provide new insights into biomass allocation as a response to PHE exposure. Indeed, in this study PHE-exposed maize seemed to preserve both foliar and root system allocation, yet producing thinner leaves, at the expense of the stem. These morphological modifications would indicate that maize compensates for PHE exposure to optimize photosynthesis: With greater leaf surface per unit biomass, plants would apparently maximize light exposure and gas-exchange for photosynthetic cells. However, measurements showed decreased transpiration and CO2 absorption in plants exposed to PHE concentrations exceeding 50 mg kg1. This reduced photosynthetic carbon fixation could be the reason for lower biomass production. Concerning the root system, root biomass allocation was significantly higher for maize exposed to PHE and was accompanied by a thickening of root system, which is in agreement with the findings of Kummerová et al. (2013). These morphological changes to the root system were observed for all PHE exposure levels. Root nutrient content was unaffected by PHE exposure, except for the highest exposure level which showed N and K depletions, and S, Mn and Fe accumulations. Preservation of leaf biomass in the case of PHE exposure was accompanied by a modification to the nutrient composition with K+ depletion, Ca2+, Mg2+, P and Zn2+ accumulations, yet no significant modifications to the contents of the other major elements. Potassium and calcium are known to be involved in the stomata’s functioning. Indeed potassium ion-release from guard cells is triggered by elevation of the intracellular Ca2+ concentration, and causes stomatal pore closing (Ward and Schroeder, 1994) as in the case of water-stress plant response. Furthermore, some symptoms (decreased biomass and CO2 input, high Mg2+ concentrations related to reduced photosynthesis) reported in the present study are found in plants subjected to water-stress (Rao et al., 1987; Tezara et al., 1999; Lawlor, 2002; Shao et al., 2008). However in this study, sand moisture was the same for each treatment (80% water-holding capacity) and relative water contents in roots, stems and leaves of PHE-exposed plants showed no difference to that of the control treatment. A water-stress type response has already appeared as a symptom of PAH and/or fuel oil hydrocarbon exposure (Adam and
Table 2 Mean values ± standard errors of nutrient concentrations (lmol g1 dw) in roots of maize (Zea mays) after a 28 d cultivation in sand and nutrient solution with different phenanthrene concentrations. n is the number of replicates. For each element and each plant organ mean values with the same letter are not significantly different (p < 0.05). [PHE] initial (mg kg1 dw)
n
N
Leaves
0 50 150 250 500 750
6 3 4 4 4 4
3283 ± 269 4070 ± 242 3598 ± 125 2737 ± 728 2958 ± 564 2954 ± 636
Roots
0 50 150 250 500 750
6 3 3 3 4 3
2189 ± 142 2573 ± 150 2190 ± 202 2797 ± 164 2348 ± 149 957 ± 120
n
K
Ca
a a a a a a
6 4 4 4 4 5
413 ± 62 a 567 ± 87 a 188 ± 47 b 100 ± 10 b 87 ± 9 b 81 ± 8 b
152 ± 11 250 ± 28 243 ± 26 306 ± 36 285 ± 20 328 ± 12
b a ab a a a
a a a a a b
6 3 4 4 4 5
230 ± 25 253 ± 72 194 ± 30 123 ± 16 136 ± 19 106 ± 21
209 ± 11 161 ± 18 122 ± 21 133 ± 18 110 ± 19 200 ± 20
a abc c bc c ab
a a ab ab ab b
Mg
P
83 ± 4 c 97 ± 6 bc 96 ± 11 bc 108 ± 10 ab 118 ± 6 ab 142 ± 9 a
70 ± 10 129 ± 21 152 ± 25 189 ± 20 183 ± 29 277 ± 25
59 ± 3 40 ± 4 37 ± 9 37 ± 7 25 ± 5 43 ± 7
a ab ab ab b ab
S c bc bc ab ab a
73 ± 5 a 86 ± 9 a 85 ± 3 a 74 ± 9 a 63 ± 3 a 84 ± 13 a
90 ± 5 b 119 ± 10 a 89 ± 9 b 91 ± 10 b 83 ± 6 ab 103 ± 7 b 82 ± 2 ab 85 ± 1 ab 91 ± 16 ab 72 ± 4 b 72 ± 5 b 103 ± 4 a
Fe 2.3 ± 0.2 3.1 ± 0.5 3.6 ± 0.4 5.0 ± 1.4 2.9 ± 0.2 3.1 ± 0.2
Mn a a a a a a
4.2 ± 0.4 a 6.8 ± 0.4 ab 10.1 ± 2.2 b 6.2 ± 0.4 ab 8.4 ± 0.5 b 24.5 ± 14.4 b
0.57 ± 0.07 0.90 ± 0.08 0.71 ± 0.05 0.79 ± 0.04 0.70 ± 0.02 0.91 ± 0.11
Zn b a ab ab ab a
0.81 ± 0.11 1.14 ± 0.06 1.72 ± 0.13 1.55 ± 0.21 1.39 ± 0.09 1.72 ± 0.06
c bc a ab ab a
0.41 ± 0.06 ab 0.32 ± 0.02 ab 0.28 ± 0.0 b 0.26 ± 0.03 b 0.27 ± 0.04 b 0.60 ± 0.10 a
1.57 ± 0.32 0.97 ± 0.10 1.63 ± 0.30 1.38 ± 0.05 1.23 ± 0.21 1.43 ± 0.19
a a a a a a
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Duncan, 2002; Merkl et al., 2005; Langer et al., 2010). A plant’s defense mechanisms to water-stress can also disturb oxidant defense (Jiang and Zhang, 2002). This may be related to oxidative stress coupled with PAH exposure as reported in several studies (Alkio et al., 2005; Pašková et al., 2006; Liu et al., 2009), resulting in tissue and cell damage. Hypothetically, the PHE molecule or its downstream products may induce phytohormone abscisic acid ˇ ová et al., 2009), or interact with (ABA) molecule production (Ván ABA receptors in Zea mays, leading to water-stress responses. ABA plays a major role in this chemical signaling (Pospíšilová, 2003). Weisman et al. (2010) revealed slight correlations with PHE and ABA gene expression in A. thaliana, but showed an involvement of phyto-hormones and defense signaling pathways in response to PAHs. Consequently, PHE or its metabolites might have impacted the plant’s physiology on various levels, including its nutritional processes, as revealed by the concentration modifications to some nutrients in both leaves and roots (Osakabe et al., 2014). 5. Conclusion Phenanthrene appeared to be highly toxic to maize. Plant growth was altered when the substrate contained 50 mg kg1. However, plants showed an initial adaptive response to PHE toxicity, through a greater allocation of carbon assimilates to roots, a similar one to leaves and a lower one to stems. PHE-exposure caused the roots to be thicker and the leaves thinner. Maize plants exposed to PHE presented symptoms of the water-stress type response although the plants suffered no water supply limitation. PHE exposure modified mineral plant nutrition with increasing calcium content in leaves. Calcium is known to be involved in plant signaling and environmental stress response in terms of growth and development. These results provide a new perspective for plant growth on contaminated soil in a phytoremediation context, especially for the agronomic management of a phytoremediation process. The development of the root system seemed crucial and further investigation is needed to assess the effect of PHE exposure to the roots’ inner structure (e.g. apoplastic barriers, xylem or phloem vessels). Acknowledgement This work was financially supported by the Région Lorraine and SNOWMAN IBRACS project. The authors thank Christophe Robin for his advice and for the Li-Cor6200 machine, Romain Goudon for his help in ICP analysis, as well as Lucas Charrois and Rémi Baldos for plant biomass conditioning. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.11.051. References Adam, G., Duncan, H., 2002. Influence of diesel fuel on seed germination. Environ. Pollut. 120, 363–370. Ahammed, G.J., Yuan, H.-L., Ogweno, J.O., Zhou, Y.-H., Xia, X.-J., Mao, W.-H., Shi, K., Yu, J.-Q., 2012. Brassinosteroid alleviates phenanthrene and pyrene phytotoxicity by increasing detoxification activity and photosynthesis in tomato. Chemosphere 86, 546–555. Alexander, M., 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol. 34, 4259–4265. Alkio, M., Tabuchi, T.M., Wang, X., Colón-Carmona, A., 2005. Stress responses to polycyclic aromatic hydrocarbons in Arabidopsis include growth inhibition and hypersensitive response-like symptoms. J. Exp. Bot. 56, 2983–2994.
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