Oxidative stress links response to lead and Acyrthosiphon pisum in Pisum sativum L.

Journal of Plant Physiology 240 (2019) 152996

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Oxidative stress links response to lead and Acyrthosiphon pisum in Pisum sativum L.

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Agnieszka Woźniaka, Waldemar Bednarskib, Katarzyna Dancewiczc, Beata Gabryśc, Beata Borowiak-Sobkowiakd, Jan Bocianowskie, Sławomir Samardakiewiczf, ⁎ Renata Rucińska-Sobkowiakg, Iwona Morkunasa, a

Department of Plant Physiology, Poznań University of Life Sciences, Wołyńska 35, 60-637, Poznań, Poland Institute of Molecular Physics, Polish Academy of Sciences, Smoluchowskiego 17, 60-179, Poznań, Poland c Department of Botany and Ecology, University of Zielona Góra, Prof. Z. Szafrana 1, 65-516, Zielona Góra, Poland d Department of Entomology and Environmental Protection, Poznań University of Life Sciences, Dąbrowskiego 159, Poznań, 60-594, Poland e Department of Mathematical and Statistical Methods, Poznań University of Life Sciences, Wojska Polskiego 28, 60-637, Poznań, Poland f Laboratory of Electron and Confocal Microscopy, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614, Poznań, Poland g Department of Plant Ecophysiology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614, Poznań, Poland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lead-plant-aphid interactions Hormetic and sublethal doses Oxidative stress Antioxidant system Aphid probing behavior Demographic parameters

This study demonstrates the impact of lead at hormetic (0.075 mM Pb(NO3)2) and sublethal (0.5 mM Pb(NO3)2) doses on the intensity of oxidative stress in pea seedlings (Pisum sativum L. cv. ‘Cysterski’). Our first objective was to determine how exposure of pea seedlings to Pb alters the plant defence responses to pea aphid (Acyrthosiphon pisum Harris), and whether these responses could indirectly affect A. pisum. The second objective was to investigate the effects of various Pb concentrations in the medium on demographic parameters of pea aphid population and the process of its feeding on edible pea. We found that the dose of Pb sublethal for pea seedlings strongly reduced net reproductive rate and limited the number of A. pisum individuals reaching the phloem. An important defence line of pea seedlings growing on Pb-supplemented medium and next during combinatory effect of the two stressors Pb and A. pisum was a high generation of superoxide anion (O2%−). This was accompanied by a considerable reduction in superoxide dismutase (SOD) activity, and a decrease in the level of Mn2+ ions. A the same time, weak activity of Mn-SOD was detected in the roots of the seedlings exposed to the sublethal dose of Pb and during Pb and aphid interaction. Apart from the marked increase in O2%−, an increase in semiquinone radicals occurred, especially in the roots of the seedlings treated with the sublethal dose of Pb and both infested and non-infested with aphids. Also, hydrogen peroxide (H2O2) generation markedly intensified in aphid-infested leaves. It reached the highest level 24 h post infestation (hpi), mainly in the cell wall of leaf epidermis. This may be related to the function of H2O2 as a signalling molecule that triggers defence mechanisms. The activity of peroxidase (POX), an important enzyme involved in scavenging H2O2, was also high at 24 hpi and at subsequent time points. Moreover, the contents of thiobarbituric acid reactive substances (TBARS), products of lipid peroxidation, rose but to a small degree thanks to an efficient antioxidant system. Total antioxidant capacity (TAC) dependent on the pool of fast antioxidants, both in infested and non-infested and leaves was higher than in the control. In conclusion, the reaction of pea seedlings to low and sublethal doses of Pb and then A. pisum infestation differed substantially and depended on a direct contact of the stress factor with the organ (Pb with roots and A. pisum with leaves). The probing behavior of A. pisum also depended on Pb concentration in the plant tissues.

1. Introduction Under natural conditions, many stress factors often act simultaneously or successively. Plants are exposed to a wide range of pollutants

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found in the environment at varying concentrations and toxicity (Pourrut et al., 2011). Studies on the responses of organisms, including plants and insects, to various concentrations of pollutants at the hormetic and toxic levels are important from the perspective of ecology

Corresponding author. E-mail addresses: [email protected], [email protected] (I. Morkunas).

https://doi.org/10.1016/j.jplph.2019.152996 Received 14 December 2018; Received in revised form 8 June 2019; Accepted 11 June 2019 Available online 13 June 2019 0176-1617/ © 2019 Published by Elsevier GmbH.

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concentration. The effects of Pb on pea have been documented in literature (Małecka et al., 2009, 2008). However, there are no data concerning induction of oxidative stress defence response and activation of the antioxidant system during an experimental sequence involving an abiotic factor, such as Pb, followed by Pb and a biotic factor such as A. pisum, an insect with a piercing-sucking mouthpart. Studies published so far indicated that in the urban environment (roadside trees) phytophages with sucking mouth organs (aphids, Psylloidea, Auchenorrhyncha and thrips) typically predominate over species with a chewing mouthpart that prefer less contaminated locations in housing districts (Lubiarz et al., 2011). Additionally, earlier results showed much gretaer numbers of aphid species in the urban environment than in uncontaminated areas (Lubiarz and Cichocka, 2003). Insects play a welldefined role in the trophic chain and serving as food for other organisms they may constitute an important path for bioaccumulation of heavy metals. As mentioned above, Pb is the heavy metal of limited mobility that is first accumulated mainly in the roots. At higher concentrations some of the accumulated Pb is transported to the leaves. These properties were considered when seleting this metal to tackle the proposed problem in this research. Our previous study (Woźniak et al., 2017a), carried out in the same research model, showed that high concentration of Pb in the medium, i.e. 0.5 mM Pb(NO3)2, resulted in high accumulation of this element first in the roots and then in the leaves of pea seedlings. Also, Pb was downloaded by pea aphids especially at the sublethal concentration. At low concentration, (0.075 mMPb(NO3)2) this element was mainly accumulated in the roots. Using two Pb concentrations in this study (one hormetic and one sublethal), we expected differences in the intensity of ROS and semiquinone radicals (stable radicals of organic origin) generation, in the period of their generation, in the transduction of signal induced by H2O2 from roots to leaves and in the activity of antioxidant system. Identification of the above defence responses in pea seedlings in the context of interaction between Pb stress and biotic stress imposed by the insect with the piercing-sucking mouthpart is completely novel. Our results provide also an answer to an important questions: to what degree the presence of Pb at different concentrations in the roots and then in the leaves of pea seedlings affect colonisation and feeding intensity by aphids. Therefore, our first objective was to verify whether the presence of Pb in pea seedling organs depending on the concentration of this metal would trigger oxidative stress defence responses to A. pisum (generation of free radicals such as ROS and semiquinone radicals). Additionally, the study investigated the efficiency of the antioxidant system during exposure of pea seedlings to Pb at the hormetic and sublethal levels and during combinatory action of the two stressors, i.e. Pb and A. pisum interaction. Therefore, SOD and POX activity and total antioxidant capacity (TAC) – depending on the pool of slow (tyrosine and tryptophan residues) and fast-acting (ascorbic acid or glutathione) antioxidants were determined. The study also estimated the degree of cell membrane damage caused by Pb and A. pisum infestation, expressed by the contents of TBARS, products of lipid peroxidation. The second aim was to examine the influence of Pb on demographic parameters of the A. pisum population (pre-reproductive, reproductive and post-reproductive periods, fecundity and longevity) and the feeding process by electronic recording of feeding with the Electrical Penetration Graph technique (EPG). Studies concerning the effects of pollutants, including heavy metals such as Pb, on plant defence response make it possible to predict this response in plants exposed to a wide range of biotic factors. An increasing body of evidence shows that plant defence against abiotic and biotic stress seems to follow common and/or complementary pathways of signal perception, signal transduction and metabolism. This does not imply a broad band of co-resistance to different stress types, but rather reflects a continuous cross-talk during the coevolution of plants and herbivores competing in an environment (Poschenrieder et al., 2006). These cross-talk signalling pathways regulate metabolic responses. Irrespective of the above, studies on the model system of A. pisum and P.

and plant physiology, and face challenges for recognition (Calabrese, 2014; Kaiser, 2003). By applying various concentrations of heavy metals, i.e. low – enhancing metabolic state of plants (priming) and toxic – negatively affecting plant growth and physiological processes, it is possible to determine differences in the nature of the plant defence response to feeding by phytophages, including insects such as aphids. Moreover, such studies expand our knowledge on the development, fecundity and behavior of insects under various concentrations of pollutants. As reported by Calabrese and Mattson (2017), the hormetic dose response has been the object of considerable research interest over the past two decades. In the opinion of those authors, median maximal hormetic stimulatory response (in plants, animals and microbes) of biphasic dose-response relationships increases in value with a growth in the number of stimulatory doses administered below the estimated threshold/zero equivalent point, i.e. the dose where the response crosses the control group value). It is known that in terrestrial ecosystems heavy metals, including Pb, mainly enter into the plants from soil, but may also come from the external atmosphere surrounding the plants. Ashraf et al. (2017) reported that recent rates of soil contamination with various heavy metals, including Pb, and their introduction into the agro-ecosystems and transfer to organisms via the food chain is an alarming situation observed on a global scale (Abrahams, 2002; Anjum et al., 2016a, 2016b). Contents of heavy metals in plant tissues affect crop productivity and grain quality. Interest in heavy metal deposition and their environmental effects, especially on ecological communities, has significantly increased recently (Baker et al., 2010; Morkunas et al., 2018; Osiadacz and Hałaj, 2016). Soils with the highest heavy metal contamination levels are found in arable fields in the vicinity of steel works. Main sources of anthropogenic Pb include zinc (Zn), Pb and copper (Cu) works and locations where these metals are mined, the industry connected with the production of batteries, sulfuric acid, cable tubes, paints, glass and crystal glass, welding materials, bearings, printing types, casings for nuclear reactors and radiation protection as well as aviation gasoline. Surface waters may also be contaminated with Pb due to the use of nitrogen fertilisers containing this metal (Krzywy et al., 2010), and the use of pesticides, fertilizers, municipal and compost wastes, as well as heavy metals released from smelting industries and metallic ferrous mines (Yang et al., 2005). According to Ai et al. (2018), plants have developed a wide range of tolerance mechanisms activated in response to Pb exposure. Contrary to other mobile elements, Pb is accumulated in the roots and may be transported to leaves after its threshold level is exceeded. Roots have developed various mechanisms to reduce Pb uptake and its transfer to the aboveground parts, and to limit its deleterious effects (Fahr et al., 2013). It is well-documented that Pb strongly affects plant growth and development, their morpho-physiological features, plant metabolism and then the crop yield (Ashraf et al., 2015; Sharma and Dubey, 2005). Accumulation of this element may cause various disturbances in plant metabolism including elevated production of reactive oxygen species (ROS) (Gill and Tuteja, 2010; Iqbal et al., 2015; Kumar et al., 2012; Manoj and Padhy, 2013; Pourrut et al., 2011; Shahid et al., 2015, 2011; Singh et al., 2010; Yadav, 2010). Plants experience oxidative stress (OS) upon exposure to heavy metals, including Pb. For example, Singh et al. (2017) proved that Pb markedly decreased growth and induced oxidative stress in Vigna radiate by enhancing the production of ROS. The OS level in plants treated with heavy metals depends on the metal mobility and bioaccessibility (Fryzova et al., 2018), and the antioxidant system ability to scavenge oxidized molecules (Clemens, 2006; Lopes et al., 2016; Rucińska-Sobkowiak, 2010). The presented research concerning mutual interactions between pea and insects may be significant, particularly in the aspect of using aphids as bioindicators of environmental changes. The culture experiments are conducted on a crop plant, i.e. edible pea that belongs to Fabaceae family. The species a wide range of applications due to high seed protein content and amino acid composition. The above model was used to indicate the differences in the plant response sequence depending on Pb 2

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7.8) containing 0.05% NBT and 10 mM NaN3, NADPH in a final volume of 3.5 mL and incubated for 1 h at room temperature. After incubation, 2 mL of the reaction solution were heated at 85 °C for 15 min and rapidly cooled. The levels of O2•− were expressed as absorbance at 580 nm per 1 g of fresh material (A580 g−1FW). The measurements were carried out using Perkin Elmer Lambda 15 UV–vis spectrophotometer (Norwalk, CT).

sativum L. are significant also to assess the effect of global climate change on living organisms, since mobility of contaminants and the rate of aphid reproduction will be changing. 2. Materials and methods 2.1. Plant material and growth conditions Pea (Pisum sativum L. cv. ‘Cysterski’) seeds of S-elite class used in the experiments were obtained from the Plant Breeding Company in Tulce near Poznań, Poland. The seed surface was sterilized as described by Mai et al. (2014) and Morkunas et al. (2016). After 6 h of imbibition the seeds were transferred onto filter paper (in Petri dishes) and immersed in small amount of water to support further absorption. After further 66 h the seed coats were removed from the germinating seeds. Next, the germinating seeds (n = 35) were transferred to hydroponic growth boxes containing Hoagland medium (the control). The boxes were covered with dark foil to mimic soil conditions. The experiment involved the seedlings of Pisum sativum L. For the first four days the seedlings were kept in the hydroponic cultures in a phytotron at 22–23 °C, 65% relative humidity and light intensity of 130–150 μmol photons m−2s-1 with 14/10 h (light/dark) photoperiod. On the fifth day the medium was replaced and Pb was added at 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 (Woźniak et al., 2017a). After the next four days, pea seedlings were infested with pea aphids. The experimental variants were as follows: control pea seedlings cultured without Pb and not colonised by pea aphids (Acyrthosiphon pisum), pea seedlings growing in Hoagland medium with varied concentrations of Pb, i.e. 0.075 mM Pb (NO3)2 and 0.5 mM Pb(NO3)2, pea seedlings growing in Hoagland medium with varied concentrations of Pb and colonised by pea aphids A. pisum, and pea seedlings growing in Hoagland medium colonised by pea aphids A. pisum. Samples for analyses were collected four days after Pb administration and prior to transferring the aphids onto pea seedlings (at 0 h), and then after 24, 48, 72 h of both stress treatments. Hydroponic cultures were aerated with an aeration system. Pea seedlings, both the control and Pb treated and the seedlings growing in the presence of Pb for colonisation by A. pisum, were cultured in glass aquariums (30 cm × 22 cm × 28 cm) and protected with gauze. The experimental treatments with the insects comprised leaves of pea seedlings, while analyses were also performed for roots, in order to obtain comprehensive information on root-leaf dependencies after the addition of Pb to the medium. The study will show to what extent Pb at different concentration in the root modulates the leaf response to A. pisum. It will also reveal whether A. pisum feeding on shoots affects or modulates the level of oxidative stress in the roots of pea seedlings. The experiment involved only adult insects (Fig. 1).

2.4. Determination of hydrogen peroxide concentration Concentration of hydrogen peroxide (H2O2) was determined following the spectrophotometric method described by Becana et al. (1986). Leaves and roots of pea (0.50 g fresh weight, FW) were homogenized with 3 mL of 5% trichloroacetic acid (TCA) and 0.10 g of activated charcoal. The homogenate was filtered through four layers of miracloth and centrifuged at 12,000×g for 30 min at 4̊C. The reaction mixture contained the extracted solution, 100 mM potassium phosphate buffer (pH 8.4) and the reagent containing 0.6 mM 4-(-2pyridylazo) resorcinol and 0.6 mM potassium-titanium oxalate in 1:1 proportion. The absorbance was measured at 508 nm (A508) using Perkin Elmer Lambda 15 UV–Vis spectrophotometer (Norwalk, U.S.), and decrease in absorbance was calculated. The reference values were obtained using 5% TCA to replace the extracted solution in the mixture. Content of H2O2 was determined from the difference of A508 between each sample and the reference solution. In order to plot the standard curve, hydrogen peroxide (30%, Sigma–Aldrich, U.S.) was diluted to the concentration range of 0.5–20 μM. The amount of hydrogen peroxide in pea leaves was expressed as nmol H2O2 g−1 FW 2.5. Detection of H2O2 generation Generation of H2O2 in plant tissues was detected by staining with a specific fluorescence agent, 20,70-dichlorofluorescein diacetate (DCFHDA, Sigma-Aldrich, U.S.), following the method of Mai et al. (2013). DCFH-DA is non-fluorescent, passively crosses the membrane of living cells and it is oxidized by H2O2 to a fluorescent dye. The fluorescence is proportional to the intracellular H2O2 level. The fresh leaves and aphids were submerged in 4 μM DCFH-DA dissolved for 12 h in 3 mM dimethylsulfoxide (DMSO) in 50 mM potassium phosphate buffer, pH 7.4. The leaves were washed twice with the loading buffer and then examined using a Zeiss LSM 510 confocal microscope (Jena, Germany). An argon laser was used for excitation at 488 nm, with emission at 565–615 nm following background subtraction. Microscope, laser and photomultiplier settings were held constant during the experiment in order to obtain comparable data. The aphids were observed using a SteREOLumar V12 fluorescence stereomicroscope (Zeiss, Göttingen Germany) with a filter set No. 38, (Excitation: BP 470/40, Emission: BP 525/50), and photographed using a digital camera (AxioCam MRc5, Zeiss, Göttingen, Germany). Confocal images were analyzed using the LSM Image Browser software, version 4.2., while fluorescence stereomicroscopic images were analyzed using the AxioVision software, version 4.8. The generation of H2O2 under a confocal- microscope and a stereomicroscope was examined in four leaves of five plants. Each experiment was repeated three times.

2.2. Aphids and infestation experiment Acyrthosiphon pisum (Harris), originally cultured and supplied by the Department of Entomology, Poznań University of Life Sciences, Poland, was reared on Pisum sativum L. in a growth chamber under conditions specified above. Eleven days after imbibition, i.e. on the ninth day of growth in Hoagland medium, the seedlings were infested with 20 apterous adult females of A. pisum. The aphid populations were monitored throughout the experiments and newborn nymphs were removed as they appeared (Mai et al., 2013; Morkunas et al., 2016). The control pea seedlings were cultured with no addition of Pb and were not colonised by pea aphids (Acyrthosiphon pisum).

2.6. Determination of semiquinone radicals and manganese ions Radicals were detected directly in pea leaves and roots using the electron paramagnetic resonance (EPR) technique (Bednarski et al., 2010; Formela et al., 2014; Mai et al., 2013; Morkunas et al., 2008; Morkunas and Bednarski, 2008). Samples of 1 g fresh weight of pea leaves were frozen in liquid nitrogen and lyophilized in a Jouan LP3 freeze dryer. The lyophilized material was transferred into EPR-type quartz tubes (diameter 4 mm). Electron paramagnetic resonance was measured at room temperature with a Bruker ELEXSYS X-band spectrometer (Rheinstettenstate, Germany). The EPR spectra were recorded

2.3. Determination of superoxide anion radical Determination of superoxide anion (O2•−) content in biological samples was based on its ability to reduce nitro blue tetrazolium (NBT) (Doke, 1983), as modified by Mai et al. (2013). Leaves and roots of pea (500 mg) were immersed in 10 mM potassium phosphate buffer (pH 3

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Fig. 1. Experimental design.

as the first derivatives of microwave absorption. Microwave power of 2 mW and a 2 G magnetic field modulation were applied in all the experiments to avoid signal saturation and deformation. EPR spectra were recorded for free radicals and Mn2+ in the magnetic field range of 3300–3360 G and with 4096 data points. To determine the number of paramagnetic centres in the samples, the spectra were double-integrated and compared with the intensity of the standard Al2O3:Cr3+ single crystal with a known spin concentration (Bednarski et al., 2010; Formela et al., 2014; Mai et al., 2013; Morkunas et al., 2013, 2008, 2004, 2003; Morkunas and Bednarski, 2008). Some background corrections of the spectra were introduced before and after the first integration to obtain a reliable absorption signal before the second integration. Concentrations of semiquinone radicals were calculated as the number of spins per 1 g of dry weight sample and the concentration of manganese ions as the number of manganese ions per 1 g of dry weight sample. In the leaves of pea sedllings, free radicals give a strong signal with g-value, 2.0028( ± 0.0005) and weak signal with g-value 2.0050( ± 0.0005). The exception are signals from variants: 0.5 mMPb (NO3)2 0 h, 0.5 mMPb(NO3)2 24 h, 0.5 mMPb(NO3)2+aphids 24 h, 0.5 mMPb(NO3)2 48 h, 0.5 mMPb(NO3)2+aphids 48 h, 0.5 mMPb (NO3)2 72 h, 0.5 mMPb(NO3)2+aphids 72 h, where signal with g-value

2.0050 has an average intensity. The width of the EPR signal was 7.7–10.4 ( ± 0.5) Gs. In the roots of pea sedllings, free radicals give a strong signal with g-value 2.0050( ± 0.0005) and a very weak signal with g-value 2.0028( ± 0.0005). The width of the EPR signal was 8.2–9.6( ± 0.5) Gs. 2.7. Extraction and assay of superoxide dismutase (SOD) and native gel electrophoresis Enzyme analyses Frozen leaves and roots (0.50 g) were homogenized at 4 °C in 2.0 mL 50 mM sodium phosphate buffer (pH 7.0) containing 1.0 mM EDTA, 2% NaCl and 1% PVP (polyvinyl pyrrolidone) and centrifuged at 15,000 × g for 15 min. The activity of SOD (EC 1.15.1.1) was spectrophotometrically assayed by measuring the enzyme ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) according to Beauchamp and Fridovich (1971), as described by Morkunas and Bednarski (2008). The reaction mixture (3 mL) contained 50 mM sodium phosphate buffer (pH 7.8), 13 mM methionine, 75 mM NBT, 0.1 mM EDTA, 30 μL of enzyme extract and 2 mM riboflavin (introduced to the reaction mixture as the last reagent). The reaction was started by switching on the light (two 15 W fluorescent lamps placed 4

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aliquots of the supernatant were mixed in test tubes with 0.5 mL of potassium phosphate buffer (pH 7.0), and 1 mL of the reagent (0.5% TBA in 20% TCA). The reference consisted of 0.5% TBA, buffer and the reagent. The test tubes were incubated at 95 °C for 30 min and then quickly cooled in an ice bath. After cooling, the mixtures were centrifuged at 10,000×g for 10 min to obtain a clear supernatant. The supernatant absorbance was measured at 532 nm and 600 nm using Jasco V-530 UV/VIS Spectrophotometer, series B225360512 (Japan). Results are presented as μmol TBARS g−1FW.

30 cm below test tubes) and proceeded for 15 min. The amount of the enzyme that caused a 50% inhibition of NBT reduction was adopted as a unit of SOD activity. The activity of the enzyme was expressed as units per 1 mg of protein (U·mg−1 protein). The protein in the samples was determined according to Bradford (1976), using bovine serum albumin (Sigma-Aldrich, U.S.) as a standard. Moreover, leaves of six to seven plants from each experimental variant were used to determine SOD activity. Each experiment was repeated three times. 2.7.1. Electrophoretic procedure Roots and leaves of pea seedlings were homogenised on ice with a mortar and pestle in 50 mM Tris−HCl buffer (pH 7.5), using 2 mL buffer per gram of fresh tissue. The slurry was centrifuged for 20 min at 15,000 g at 4 °C. Supernatant samples (30 μg protein) were electrophoresed according to Davis (1964) in 10% (w/v) polyacrylamide slab gel at pH 8.9 under non-denaturing conditions and in discontinuous buffer system..The SE 250 Mighty Small II (Hoefer) cooled miniature vertical slab gel unit was used for electrophoresis. Protein separation was accomplished as soon as the dye front came out from the bottom of the gel. Isoenzymes of SOD (EC 1.15.1.1) were visualized on gels using the photochemical procedure of Beauchamp and Fridovich (1971). The gel was first soaked in 2.45 mM NBT nitroblue tetrazolium (solution A) for 20 min, followed by 25 min immersion in a solution containing 28 mM EDTA, 20 μM riboflavin and 36 mM potassium phosphate buffer (pH 7.8) (solution B), and subsequently illuminated for 10 min (Rucińska et al., 1999). To identify the type of SOD, the gel was incubated in the absence or presence of KCN or H2O2 (Asada et al., 1975). When KCN was used as SOD inhibitor, the gel was soaked in the solution A for 20 min and immersed for 25 min in the solution B supplemented with 2 mM KCN. In the case of H2O2, the gel was incubated in 3 mM H2O2 (water solution) prior to NBT staining in solutions A and B. Moreover, it is known that Cu,Zn-SODs are generally 32 kDa homodimers, whereas Fe or Mn-SODs are 45 kDa homodimers (Miller, 2012).

2.11. Determination of the effect of lead on the demographic parameters of A. pisum population During the first four days pea seedlings were cultured in hydroponic cultures in Hoagland medium. On the fifth day the medium was replaced in all the variants and Pb was added at a concentration of 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2. Four days after the administration of Pb, (8-day-old pea seedlings)30 female aphids (apterous individuals of the same age) were individually placed on pea seedling shoots. Larvae born by these females were the material for further observations. The survival rate of larvae was determined for a population of 100 larvae. In control conditions 100% of the larvae reached maturity and at 0.075 Pb(NO3)2 and 0.5 mMPb(NO3)2 approx. 99% and 95%, respectively. Forty females were observed to calculate demographic parameters of the population. The length pre-reproduction, reproduction, and postreproduction developmental stages, and the total life span and female fecundity were recorded. The observations were carried out daily. Demographic parameters of the aphid populations at different variants (control, 0.075 mM and 0.5 mMPb(NO3)2) were determined following the methods described by Birch (1948): intrinsic rate of increase, rm; net reproductive value, Ro; finite rate of increase, λ; and mean generation time, T. The intrinsic growth rate (rm) was calculated using the formula of Wyatt and White (1977) rm = 0.738 [(lnMd)/d], where d is the development period from birth to the beginning of the first reproduction and Md is the number of nymphs born in the period from time d. Rearing was carried out in a controlled environment of a growth chamber at 22–23 °C, 65% relative humidity and light intensity of 130–150 μM photons m−2s-1 with the 14/10 h (light/ dark) photoperiod. The demographic parameters were calculated using the DEMOGRAF program (Kozłowski et al., 2010).

2.8. Extraction and assay of peroxidase (POX) activity Frozen pea seedling leaves and roots (400 mg) were homogenised at 4 °C with a mortar and pestle in 3 mL of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA and 10% (w/v) Polyclar AT, and centrifuged at 15,000 g for 30 min at 4 °C, with the supernatants used for enzyme assays. The protein concentration in the samples was estimated according to Bradford (1976). Peroxidase (EC 1.11.1.7) activity was measured spectrophotometrically using syringaldazine as a phenolic substrate, as described by Morkunas and Gmerek (2007). The activity of the enzyme was expressed as enzyme unit per 1 mg protein (U mg−1 protein).

2.12. The effect of lead on behavioral responses of A. pisum during probing and feeding Feeding behaviour of pea aphids was analysed four days after the administration of Pb (i.e. in eight-day-old pea seedlings). The probing behavior of A. pisum was investigated by means of Electrical Penetration Graph technique (EPG) (Tjallingii, 2006; Will et al., 2007). This technique is commonly applied in Hemiptera-plant relationship studies (Tjallingii, 2006; Woźniak et al., 2017b). The system is based on an electrical circuit where electrodes are attached to the aphid and the plant. When aphid stylets pierce into plant tissues, the circuit is completed. Aphid activity causes changes in the electrical properties of the circuit and these changes are manifested as EPG waveforms. Presently, the meaning of most of the waveforms is known, so it is possible to relate them to a specific aphid activity. A one hour starvation period after the attachment of the electrode was maintained before the start of the experiment. Each freshly prepared aphid/plant combination was used as an individual replicate. A completely randomized design was used for these experiments. The Giga-8 DC EPG system with a 1 GΩ of input resistance (EPG Systems, Wageningen, the Netherlands) was used to record EPGs. EPGs were recorded and analyzed using the Stylet + release 2011_5 software (EPG Systems). The EPG recording started at 10–11 a.m. and was carried out for 8 h. Various behavioral phases were labelled manually using the Stylet + software. The analysis of EPG recordings included: np (non-probing, i.e. aphids with stylets

2.9. Extraction and assay of total antioxidant capacity Determination of total antioxidant capacity was based on a reduction of cation-radical ABTS•+ to colorless ABTS as decribed by Morkunas et al. (2016, 2013). The decrease in absorbance is proportional to the amount of antioxidants present in the sample. The decrease in absorbance measured after 10 s detects typical antioxidants such as ascorbate and glutathione that react rapidly with ABTS•+. The decrease in absorbance after 30 min detects residues of tyrosine and tryptophan in proteins that react slowly with ABTS•+. The final result of TAC was expressed as micromolar Trolox per gram of fresh weight. 2.10. Lipid peroxidation Lipid peroxidation was determined by thiobarbituric acid reactive substances (TBARS) assay following minor modification from the method of Heath and Packer (1968). Frozen samples of roots and leaves (0.50 g) were homogenised in 3 mL 0.5% thiobarbituric acid (TBA) and 20% TCA and centrifuged at 12,000×g for 20 min at 4 °C. Then, 0.5 mL 5

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medium (0.075 mMPb(NO3)2), O2•− generation in the leaves at 24 and 72 h post infestation (hpi) increased by 25.22% and 43.012% (in 0.075 mMPb(NO3)2 +aphids variant), respectively, in comparison with leaves of 0.075 mMPb(NO3)2 variant, and it was also higher than in the control. Also, at low Pb concentration, an increase in O2•− was observed in the roots of pea seedlings infested by aphids at 24 and 48 hpi, as compared with non-infested seedlings. Moreover, both in the leaves and roots of the seedlings exposed to 0.075 mM Pb(NO3)2, generation of this ROS was considerably lower than in the organs of seedlings cultured with 0.5 mM Pb(NO3)2. Aphid feeding alone (+aphids variant) caused no considerable changes in O2•− level in the roots at 24 h and 48 h hpi (Fig. 2a). At 24 and 72 h hpi, O2•− level in the leaves of seedlings colonised by aphids (+aphids) was by 27.64% and 21.04% lower, respectively, than in the control. At 72 hpi, the roots of pea seedlings infested by aphids and grown at low Pb dose showed a decrease in O2•− level in relation to 0.075 mM Pb(NO3)2 variant.

outside plant tissues), C (probing in non-phloem tissues, which includes pathway C, i.e. probing in the epidermis and mesophyll, the so-called “derailed” stylet activities F, and xylem sap consumption G), E1 (salivation into sieve elements), and E2 (phloem sap consumption). Several parameters related to the sequence and frequency of aphid activities during probing and stylet position in plant tissues were analysed (Helden and van Tjallingii, 1993). Waveform patterns that were not terminated before the end of the experimental period were included in the calculations. In sequential parameters, the duration of the period preceding the first phloem phase or the first sustained ingestion phase equalled the time from the first probe until the end of the recording if either phase was not observed in a given aphid/viviparous apterous females (VIP) replicate. In sequential parameters, when time to waveforms related to phloem phase was calculated, and no phloem phase was recorded, the time from the first probe until the end of the recording was used. In the non-sequential parameters that described general aphid behavior, when a given waveform was not recorded for an individual, the duration of that waveform was given the value “0”. The phloem phase index was calculated as the phloem phase/phloem phase + non-phloem probing phase: E/(E + C + F + G); The phloem salivation index was calculated as the phloem salivation/phloem phase: E1/(E1 + E2). EPG parameters were calculated manually and individually for every aphid, while the mean and standard errors were subsequently calculated using the EPG analysis Excel worksheet created for this study. Mann–Whitney U test was applied to analyze differences in the parameters derived from EPG recordings.

3.2. The effect of lead and A. pisum on the generation of hydrogen peroxide in pea seedlings The level of H2O2 in the roots generally increased with time (Fig. 3A). However, at 24 h of the experiment the highest generation of H2O2 was observed in the roots of seedlings cultured at high concentration of Pb and infested by aphids, while at 48 h it was the highest in the roots exposed to the higher concentration of Pb. Moreover, the highest generation of H2O2 during the experiment was recorded in 72 h roots growing in the medium with a low dose of Pb and infested by A. pisum (0.075 mM Pb(NO3)2+aphids variant). In this variant, the level of H2O2 was by 78% higher than in 0.075 mM Pb(NO3)2 variant. Four days after the application of Pb, i.e. at 0 h of the experiment the leaf level of hydrogen peroxide for both 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 was over 1.5-fold higher than in the control (Fig. 3B). Next, 24 h after A. pisum colonisation a considerable increase was recorded for H2O2 levels in the leaves of pea seedlings growing in Hoagland medium. However, the highest H2O2 level was found in 24 h leaves of pea seedlings treated with 0.5 mM Pb(NO3)2 and infested by aphids (0.5 mM Pb(NO3)2+aphids variant) (Fig. 3B). The leaves exposed to low Pb dose and those exposed to low Pb dose and infested by aphids (0.075 mM Pb(NO3)2 and 0.075 mM Pb(NO3)2+aphids variants), the content of H2O2 amounted to 552.29 - 625.56 nmol·g−1 FW between 24 and 72 h hpi. Interestingly, pea aphid infestation caused no incerase in H2O2 generation in leaves vs. 0.075 mM Pb(NO3)2 variant.

2.13. Statistical analysis All determinations were conducted within three independent experiments. Additionally, three biological replicates per experimental variant were performed for a given experiment. Analysis of variance (ANOVA) was used to verify the significance of means from independent experiments within a given experimental variant. The elementary comparisons between particular levels of the analysed factor in different times (independently) were tested using a two-sample t-test for equal means for all the observed traits. To account for multiple testing we used the Bonferroni correction. Comparisons involve the following plant material variants, i.e. control vs. 0.075 mM Pb2+; control vs. 0.5 mM Pb2+; control vs. aphids; control vs. 0.075 mM Pb2++aphids; control vs. 0.5 mM Pb2++aphids; 0.075 mM Pb2+ vs. 0.5 mM Pb2+; 0.075 mM Pb2+ vs. 0.075 mM Pb2++aphids; 0.5 mM Pb2+ vs. 0.5 mMPb2++aphids; 0.075 mM Pb2++aphids vs. 0.5 mM Pb2++aphids; aphids vs. 0.075 mM Pb2++aphids; aphids vs. 0.5 mM Pb2++aphids. The figures present data as means of triplicates for each variant along with standard errors of mean (SE). All the analyses were conducted using the GenStat v. 17 statistical software package.

3.2.1. Aphid infestation increases hydrogen peroxide accumulation in epidermal cells Observations of pea seedling leaves under a confocal microscope revealed generation of H2O2 in the epidermal layer. In the control material, fluorescence indicating the presence of H2O2 was reported only sporadically in guard cells, and occasionally in cells adjacent to the guard cells. An intense green fluorescence was evident in the stomata and in adjacent cells infested by aphids, i.e. both in the + aphids variants and in 0.075 mM Pb(NO3)2+aphids and 0.5 mM Pb (NO3)2+aphids variants (Fig. 3C). Moreover, strong generation of hydrogen peroxide was observed mainly in the cell walls, while green fluorescence indicating its generation was also detected in the cytoplasm exposed to the effect of both stress factors. A particularly high fluorescence level was recorded after 24 h in the leaves from the variants + aphids, 0.075 mM Pb(NO3)2+aphids, 0.5 mM Pb (NO3)2+aphids, and 0.5 mM Pb(NO3)2.

3. Results 3.1. The effect of lead and A. pisum on the generation of superoxide anion radical in pea seedlings Four days after Pb application, i.e. at 0 h of the experiment, the amount of superoxide anion radical (O2•−) both in the leaves and roots of pea seedlings growing in the medium with sublethal (0.5 mM Pb (NO3)2) dose of Pb was over 1.5 and 4-fold greater than in the control, respectively (Fig. 2). Additionally, at 0 h of the experiment, the level of O2•−in the leaves from 0.075 mM Pb(NO3)2 variant was by 24.79% lower than in the control and it showed a different trend than in the roots. High level of this ROS was maintained until the next time point, i.e. 24 h, both in the leaves and roots growing in the medium with 0.5 mM Pb(NO3)2. Aphid feeding on the leaves exposed to 0.5 mM Pb (NO3)2 (0.5 mMPb(NO3)2+aphids variant) reduced O2•− level as compared with the leaves from 0.5 mM Pb(NO3)2 variant, but it was still higher than in the control (Fig. 2b). At low Pb concentration in the

3.2.2. The effect of lead on relative generation of hydrogen peroxide in aphid body Irrespective of the above, at 72 hpi on pea seedlings growing with varied Pb concentrations, generation of H2O2 was observed in the aphid body, being much higher than in the control aphids (Fig. 3D). In the control aphids (without Pb), fluorescence was visible but only in a small 6

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Fig. 2. The effect of lead and A. pisum on the content of superoxide anion in the roots (a) and leaves (b) of pea seedlings. The data were obtained in three independent experiments and statistically analyzed using ANOVA (p-values at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1).

(+aphids variant) contained lower amounts of these ions than the control seedlings. Moreover, 24 and 72 hpi Mn2+ levels in the leaves of pea seedlings from the + aphids, 0.075 mM Pb(NO3)2, and 0.075 mM Pb(NO3)2+aphids variants were comparable to those in the control leaves (Fig. 4D). Higher level of these ions than in control was observed only in the leaves 48 hpi in the above variants. In the leaves exposed to the toxic dose of Pb, both infested and non-infested by A. pisum, the levels of Mn2+ at 24 and 72 hpi were lower than in the control and in the other stress variants.

area, on the margins of the dorsal part of the thorax. In the case of aphids feeding on Pb-treated plants the level of H2O2 increased markedly. Fluorescence was observed in those insects both on the dorsal and abdominal side of the whole thorax and the abdomen. In the case of the variant with the higher Pb concentration fluorescence was also observed in the aphid legs. 3.3. The effect of lead and A. pisum on semiquinone radical generation and manganese ions in pea seedlings

3.4. The effect of lead and A. pisum on the antioxidant enzyme activity in pea seedlings

Concentration of semiquinone radicals in organs of pea seedlings four days after Pb supplementation, i.e. at 0 h of the experiment, was generally higher than in the control seedlings (Fig. 4). At this time point, the recorded level of semiquinone radicals in the roots of pea seedlings treated with 0.5 mM Pb(NO3)2 was two times greater than in the control roots (Fig. 4A). At successive time points, i.e. at 24 and 72 h of the experiment the level of these radicals remained high in the roots exposed to the toxic Pb dose, i.e. in 0.5 mM Pb(NO3)2 and 0.5 mM Pb (NO3)2+aphids variants. The concentration of these radicals in the roots from the above experimental variants ranged from 2.21·1015 radicals·g−1dry weight (DW) to 4.19·1015 radicals g-1DW and it was markedly higher than in the other experimental variants between 24 to 72 hpi. Until 24 h of the experiment, roots exposed to the low dose of lead (0.075 mM Pb(NO3)2 and 0.075 mM Pb(NO3)2+aphids variants) showed the level of these radicals comparable to the control. Only at 48 hpi, their concentration decreased as comapred with the control. The trends in the level of semiquinone radicals differed in pea leaves and roots (Fig. 4). A. pisum infestation on pea seedlings (+aphids variant) and the combinatory effect of the two stressors, i.e., a low dose of Pb (0.075 mM Pb(NO3)2) and A. pisum, considerably enhanced the level of semiquinone radicals in relation to the control seedlings evaluated at 48 hpi to 72 hpi (Fig. 4B). Additionally, the concentration of these radicals in the leaves exposed to low level of Pb (0.075 mM Pb(NO3)2 variant) at 24 and 72 h of the experiment was similar. For the toxic Pb concentration at 48 hpi the level of semiquinone radicals in the leaves was lower than in the leaves of the seedlings growing in the presence of low Pb dose. EPR spectroscopy revealed that at 0 h of the experiment, i.e. four days after lead administration, the content of Mn2+ in the roots and leaves of pea seedlings treated with Pb decerased as compared with the control (Fig. 4). The decrease was significantly larger for the sublethal dose of Pb. At successive time points, i.e. from 24 to 72 h of the culture, the roots exposed to low and toxic concentrations of Pb showed a lower concentration of Mn2+ than the controls (Fig. 4C). Additionally, between 48 and 72 hpi the roots of the seedlings infested by aphids

3.4.1. The effect of lead and A. pisum on superoxide dismutase activity in pea seedlings At 0 h of the experiment, i.e. after four days of pea seedling culture in the presence of 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 the activity of superoxide dismutase (SOD) decreased in relation to the control (Fig. 5). Moreover, a considerable drop in SOD activity in the organs of seedlings in all the experimental variants vs. control was observed also at successive time points from 24 to 72 h, except for 48 h and 72 h leaves of the + aphids variant. Moreover, SOD activity in pea roots (Fig. 5A) was much greater than in the leaves (Fig. 5B). Three SOD isozymes were detected in native PAGE (Fig. 5C and D). Gel incubation in KCN and H2O2 preceding staining to reveal SOD activity showed that the first isozyme was insensitive to both inhibitors, which made it possible to identify it as Mn–SOD, while the second and third isozymes were sensitive to both inhibitors, each being Cu,Zn–SOD. Electrophoretic analysis showed low content of Mn-SOD isozyme in the roots of pea seedlings growing in the presence of 0.5 mM Pb(NO3)2 and exposed to 0.5 mM Pb(NO3)2 and aphid interaction. 3.4.2. The effect of lead and A. pisum on peroxidase activity in pea seedlings Four days after the administration of Pb at the toxic concentration peroxidase activity towards a phenolic substrate, i.e. syringaldazine (POX), in the leaves of pea seedlings was five times greater than in the control (Fig. 5D). In the roots treated with 0.5 mM Pb(NO3)2, the enzyme activity showed different trends than in the leaves (Fig. 5D). Transfer of aphids onto pea seedlings generally enhanced POX activity as compared with control. However, the greatest activity of the enzyme in the leaves, i.e. 9.42 U· mg−1 protein, was recorded at 48 hpi in 0.5 mM Pb(NO3)2+aphids variant. In the leaves treated with 0.075 mM Pb2+ +aphids, the activity of POX was 1.95–2.08 U· mg−1 protein, and 7

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(caption on next page)

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Fig. 3. The effect of lead and A. pisum on the concentration of hydrogen peroxide in the roots (A) and leaves (B) and relative generation and cytochemical localization of hydrogen peroxide in the leaves (C) of pea seedlings. The effect of lead on relative generation of hydrogen peroxide in aphid body at 72 hpi (D). Green fluorescence originating from DCFH-DA (dichlorodihydro-fluorescein diacetate) was observed under c) a confocal microscope (Zeiss LSM 510; objective magnification of 40×, scale bar 50 μm), d) fluorescence steromicroscope (Zeiss, SteREO LumarV12, objective magnification of 0,8×, scale bar 1 mm). The data were obtained in three independent experiments and statistically analyzed using ANOVA (p-values at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

3.5. The effect of lead on total antioxidant capacity dependent on the pool of slow and fast antioxidants

it was higher than in the control, but significantly lower than in 0.5 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2+aphids variants. At 24 hpi the enzyme activity in pea roots in the + aphids variant was 3.5 times greater than in the control (Fig. 5C). It was found that Pb alone applied at the toxic concentration considerably increased POX activity in the roots, particularly at 48 and 72 hpi. The enzyme activity reached then 81.67 and 54.73 U·mg−1protein, respectively and it was considerably greater than in the other experimental variants (Fig. 5C). Feeding of aphids on the seedlings cultured at the toxic Pb concentration at 24 hpi strongly enhanced POX activity as compared with other experimental variants. Moreover, POX activity in 24 h roots of pea seedlings growing in the medium with 0.075 mM Pb(NO3)2 was greater than in the control and in + aphids and +0.5 mM Pb(NO3)2 variants but lower than in 0.5 mM Pb(NO3)2+aphids variant. Only in 48 h roots treated with 0.075 mM Pb(NO3)2 and infested by aphids the recorded POX activity was greater than in 0.075 mMPb(NO3)2 variant.

Four days after the administration of toxic Pb dose, total antioxidant capacity (TAC) dependrnt on the pool of slow antioxidants in roots of pea seedlings was by 10% higher than in the control and the roots growing in the medium with 0.075 mM Pb(NO3)2 (Fig. 6A). At the subsequent time points, i.e. at 24 h and 48 h a slight increase (10–15%) in TAC dependent on the pool of slow antioxidants, was noted in the roots exposed to the toxic Pb dose both infested and non-infested by A. pisum (0.5 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2+aphids variants), as compared with the control and other experimental variants. An opposite trend was observed for TAC dependent on the pool of fast antioxidants in the roots growing in the presence of toxic Pb dose, where a reduction in TAC level was detected (Fig. 6C). Besides, in the roots of the seedlings infested by A. pisum, a slight decrease in TAC dependent on the pool of slow antioxidants vs. other experimental variants was noted. TAC dependent on the pool of fast antioxidants was similar in the control roots and the roots treated with 0.075 mM Pb(NO3)2.

Fig. 4. The effect of Pb and A. pisum on the concentration of semiquinone radical (a,b) and Mn2+ ions (c,d) in the organs of pea seedlings as detected by EPR. The data were obtained in three independent experiments and were statistically analyzed using ANOVA (p-values at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1). 9

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Fig. 5. The effect of Pb and A. pisum on superoxide dismutase activity (a, b) and peroxidase activity (c, d) in the roots (a, c) and leaves (b, d) of pea seedlings. The data were obtained in three independent experiments and statistically analyzed using ANOVA (p-values at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1).

0 and 24 h of the experiment, the leaves of the seedlings growing at hormetic and toxic Pb doses showed a higher level of TAC dependent on the pool of fast antioxidants.

TAC dependent on the pool of slow antioxidants was reduced in the leaves treated with toxic Pb dose both infested and non-infested by A. pisum (Fig. 6B). An opposite trend was observed for TAC dependent on the pool of fast antioxidants that increased at all time points in the leaves treated with toxic Pb dose both infested and non-infested by A. pisum, as compared with other experimental variants (Fig. 6D). Only at

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Fig. 6. The effect of Pb and A. pisum on total antioxidant capacity dependent on the pool of slow antioxidants in roots (a) and leaves (b) and dependent on the pool of fast antioxidants in roots (c) and leaves (d) of pea seedlings. The data were obtained in three independent experiments and statistically analyzed using ANOVA (pvalues at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1).

on the length of the reproduction period, while the higher Pb concentration shortened the reproduction period by 33% in comparison with the control. Both Pb concentrations slightly extended the postreproduction period vs. the control. No significant differences were found between the total life span of A. pisum cultured on the control pea seedlings and those cultured on the seedlings treated with the lower Pb dose. Toxic Pb dose shortened the total life span of pea aphids by 28% in relation to the control. Pb significantly reduced A. pisum fecundity (Table 1A and Fig. 8A). For the lower Pb dose (0.075 mM Pb(NO3)2) fecundity decreased by 21%, while for the higher by as much as 52% (Table 1, Fig. 8A). Analyses of the demographic indexes of the population revealed that Pb at the toxic dose significantly reduced the net reproduction rate (R°), while the effect was small for the low Pb dose (Table 1B). Lead also slightly changed rm (intrinsic rate of increase), λ (finite rate of increase) and T (generation time).

3.6. Lipid peroxidation The sublethal dose of Pb boosted TBARS content in pea leaves and roots (Fig. 7). At 0 h of the experiment, i.e. after for days of pea seedling culture in the medium with 0.5 mM Pb(NO3)2, the level of TBARS increased in relation to the control and 0.075 mM Pb(NO3)2 variant. The content of TBARS in 0-h roots of pea seedlings was five times higher and in the leaves was 2.5 times higher than in the control (Fig. 7A). High content of the lipid peroxidation product was also observed at subsequent time points in the seedling organs for 0.5 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2+aphids variants. This content was higher than in the control and other experimental variants. Closer attention was paid to the higher level of TBARS in the leaves than in the roots. Additionally, between 24–72 hpi generally higher levels of TBARS were observed in the roots of the seedlings exposed to aphids from the 0.075 mM Pb (NO3)2 and 0.075 mM Pb(NO3)2+aphids variants. TBARS contents in the leaves infested by A. pisum and leaves from 0.075 mM Pb (NO3)2+aphids variant were generally higher than in control (Fig. 7B).

3.8. The effect of lead on behavioral responses of A. pisum during probing in plant tissues The EPG recording of the probing behavior of A. pisum revealed all kinds of aphid stylet activities, which include penetration of nonphloem tissues, i.e. pathway ‘C’, short punctures of epidermal and mesophyll cells, known as ‘potential drops’ and xylem sap ingestion ‘G’, and penetration of phloem tissue, i.e. watery salivation ‘E1’ and sap ingestion ‘E2’ (Table 2, Fig. 8). Stylet penetration in plant tissues

3.7. The effect of lead on demographic parameters of A. pisum population The prereproduction period for the control and the variants with varied lead nitrate concentrations was approximately six days (Table 1A). No effect of Pb on the length of the prereproduction period was observed. The lower Pb concentration in the substrate had no effect 11

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Fig. 7. The effect of Pb and A. pisum on the content of thiobarbituric acid reactive substances (TBARS) in the roots and leaves of Pisum sativum cv. ‘Cysterski’ seedlings. The data were obtained in three independent experiments and statistically analyzed using ANOVA (p-values at α = 0.05). Hypotheses on the equality of means were verified by the two-sample t-test. To account for multiple testing, we used the Bonferroni correction (statistically significant differences are shown in Table S1).

phase in the case of aphids found on seedlings growing with Pb at the hormetic dose was higher (2.2 probes) than in the control (1.79 probes). The non-probing between probes amounted to 2.7% for A. pisum on plants treated with 0.075 mM Pb(NO3)2, 3.7% for A. pisum on plants treated with 0.5 mM Pb(NO3)2, and 6.3% on control plants (Table 2). These outcomes demonstrate that the presence of Pb shortened the total duration of the non-probing phase (np). There were minimum 1.87 (0.5 mM Pb(NO3)2) up to maximum 3.2 (0.075 mM Pb (NO3)2) phloem phases per aphid and they were mostly the periods of sustained sap ingestion (‘E2’ pattern longer than 10 min). The number

accounted for approximately 94% of experimental time on the control plants and 97% on Pb-treated plants, irrespective of Pb concentration. All aphids on all plants showed phloem sap ingestion activity (Fig. 8) that took 62% (control), 69% (0.075 mM Pb(NO3)2) and 65% (on 0.5 mM Pb(NO3)2) of the entire probing time (Table 2). On all plants the proportion of time dedicated to sap ingestion (E) increased over time, while the proportion of time spent on probing in peripheral tissues and non-probing decreased accordingly (Fig. 8). Aphid probing was rarely interrupted; there were 8.6 to 13.8 probes per aphid during the eighthour experiment. However, the number of probes with the phloem 12

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Table 1 Mean developmental time, longevity, total fecundity and rate of development of A. pisum feeding on P. sativum seedlings growing in Hoagland medium (control) and with different concentrations of lead nitrate, i.e. 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 (n = 40) (a). Hypotheses on equality of means were verified by twosample t-test (Table S1). Population and life table parameters of apterous A. pisum on P. sativum seedlings growing in Hoagland medium (control) and with different concentrations of lead nitrate, i.e. 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2. Ro, net reproductive rate; rm, intrinsic rate of increase; λ, finite rate of increase; T, generation time; DT, doubling time (b). A

Pre-reproductive period (days) Reproductive period (days) Post-reproductive period (days) Fecundity Longevity

Control

0.075 mM Pb(NO3)2

0.5 mM PB(NO3)2

6.375 ± 0.49 22.97 ± 1.25 1.37 ± 1.25 175.8 ± 8.59 32.25 ± 3.42

6.175 ± 0.38 21.575 ± 2.977 1.7 ± 2.66 138 ± 17.37 30.1 ± 3.96

6.35 ± 0.48 15.35 ± 3.34 1.425 ± 2.074 84 ± 14.48 23.25 ± 3.31

B Variants

Control 0.075 mM Pb(NO3)2 0.5 mM Pb(NO3)2

Parameter Ro

rm

λ

T

DT

67.458 58.168 31.303

0.335 0.325 0.287

1.398 1.384 1.332

12.572 12.503 11.999

2.069 2.133 2.415

4. Discussion

of sustained sap ingestion phases (E2 > 10 min) was lower than in control for the aphids on the plants treated with lower Pb dose and higher for aphids on the plants treated with higher Pb dose. Mean duration of the ingestion phase E2 (h) for aphids feeding on Pb treated plants was longer than for the control seedlings. The ingestion phase was relatively long, i.e. maximum 3.85 (higher Pb dose), and minimum 2.96 h (control plants) (Table 2). Aphids also showed activities associated with the uptake of xylem sap (pattern ‘G’). Total duration of the xylem phase for aphids on the seedlings exposed to 0.075 mM Pb(NO3)2 amounted to 7.4 min and was shorter than for aphids on the control plants (10.54 min). For the aphids feeding on plants exposed to 0.5 mM Pb(NO3)2 it was longer and lasted for 17.46 min. However, the duration of this activity for aphids from all variants was relatively short, i.e. 1.5–3.6% of the probing time (Table 2). The waveform E1 represents the ingestion of watery saliva into the sieve elements. Short periods of salivation always precede sap ingestion and are perceived as a kind of ‘preparation’ of sieve elements for prolonged ingestion of sap by aphids.

Our study shows that insect-host plant interaction can serve as a model to examine heavy metals effects on biological systems. This is the first research to reveal the effects of various Pb doses on aphid population development under controlled conditions and probing behaviour of A. pisum on edible pea (P. sativum L. cv. Cysterski). Moreover, we showed for the first time the level of oxidative stress defence responses in the organs of pea seedlings during the abiotic factor (Pb) and the biotic stress factor (A. pisum) interaction. In short, the study found that Pb at the sublethal dose for pea seedlings, i.e. 0.5 mM Pb(NO3)2, reduced fecundity and viability of aphids, which was not observed at hormetic concentration of Pb (Table 1, Table 2, Fig. 8). Moreover, as shown in our previous paper (Woźniak et al., 2017a), low Pb dose (0.075 mM Pb(NO3)2) triggered hormetic effects in pea seedlings and the groups of metabolites from this variant and from the control were clustered close to one another. EPG outcomes presented in this paper confirmed that Pb at the sublethal dose considerably extended the time required by aphids to reach the phloem (time to the first phloem phase E1, Table 2, Fig. 8). This suggests that Pb alone applied at a high,

Fig. 8. The effect of lead on fecundity of 40 apterous females of A. pisum cultured on P. sativum L. cv. ‘Cysterski’ seedlings (a). EPG recorded probing behaviour of A. pisum on Pisum sativum seedlings growing in Hoagland medium with different concentrations of lead, i.e. 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 shown as percentage of stylet activities during the eight-hour experiment. np-non penetration, C-pathway activities, G-xylem phase, E-phloem salivation and ingestion (b). Cumulative percentage of A. pisum reaching the phloem during the eight-hour EPG recording experiment conducted on Pisum sativum seedlings growing in Hoagland medium with different concentrations of lead, i.e. 0.075 mM Pb(NO3)2 and 0.5 mM Pb(NO3)2 (c). 13

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Table 2 Effect of lead on probing behaviour of A. pisum during probing in pea tissues.

General aspects of aphid probing behaviour Total duration (min) of non-probing phase (np)1 Total duration (h) of pathway phase (C)2 Total duration (min) of xylem phase (G)3 Total duration (h) of phloem phase (E1+E2)4 Phloem phase index E/(C + G+E)5 Phloem salivation index E1/(E1+E2)6 Number of probes Number of probes with phloem phase Mean duration of a probe (h) Number of phloem phases Number of sustained sap ingestion phases (E2 > 10 min) Mean duration of ingestion phase E2 (h) Activities in peripherial tissues before the first phloem phase Duration of the first probe (min) Number of probes before the first phloem phase E17 Number of probes < 3 min. before the first phloem phase7 Time to the first phloem phase E1(h)7 Time to the first sustained phloem sap ingestion E2 > 10 (h)7 Total duration of non-probing before the first E (min)7 Total duration of non-phloem probing before the first phloem phase E (min)7 Duration of the first phloem phase E1 + E2 (h)

Control n=14

0.075 mM Pb(NO3)2 n = 15

0.5 mM Pb(NO3)2 n = 15

30.35 ± 8.18 2.23 ± 0.40 10.54 ± 5.52 5.09 ± 0.57 0.7 ± 0.1 0.03 ± 0.008 13.79 ± 3.86 1.79 ± 0.24 1.16 ± 0.26 2.43 ± 0.34 2.0 ± 0.3 2.96 ± 0.66

13.11 ± 3.52 2.19 ± 0.42 7.4 ± 4.98 5.46 ± 0.49 0.7 ± 0.059 0.03 ± 0.012 8.6 ± 1.62 2.2 ± 0.38 1.99 ± 0.54 3.2 ± 0.75 2.27 ± 0.41 3.85 ± 0.85

17.94 ± 4.28 2.22 ± 0.37 17.46 ± 9.81 5.19 ± 0.46 0.7 ± 0.01 0.03 ± 0.014 8.6 ± 1.34 1.47 ± 0.17 1.27 ± 0.19 1.87 ± 0.38 1.4 ± 0.16 3.56 ± 0.63

52.51 ± 33.71 6.78 ± 2.08 4.79 ± 1.65 1.09 ± 0.16 a 1.26 ± 0.22 16.88 ± 3.63 a 53.25 ± 6.96 a

38.47 ± 31.92 3.2 ± 0.98 2.53 ± 0.8 0.67 ± 0.15 ac 0.98 ± 0.18 6.44 ± 1.60 b 35.99 ± 7.83 ac

17.99 ± 14.14 5.6 ± 1.12 3.47 ± 0.74 1.73 ± 0.4 ab 2.0 ± 0.39 13.88 ± 4.23ab 90.77 ± 20.08 ab

2.84 ± 0.74

3.67 ± 0.9

3.59 ± 0.73

n = number of replications; calculations referring to the phloem phase included only aphids that showed phloem phase. a; b; c; different letters in rows show significant differences among treatments (Mann–Whitney-U test, p < 0.05). 1 Non-probing phase np: aphid stylets withdrawn from plant. 2 Pathway with cell punctures C. 3 Xylem phase G. 4 Phloem phase includes salivation into sieve elements E1 and sap ingestion E2. 5 Index calculated as: duration of phloem phase E1 + E2/duration of phloem phase E1 + E2 + non-phloem probing phase C + F + G. 6 Index calculated as: duration of phloem salivation E1/duration of phloem phase E1 + E2. 7 All individuals were included in analysis; when an aphid did not show phloem phase, the time/number of probes to the first E1 or the first E2 was the time/ number of probes until the end of the experiment.

then infested by A. pisum was sufficient to prevent intense production of O2•−. Enhanced SOD activity in the leaves may be related to lower accumulation of Pb in these organs than in the roots, as we demonstrated in another paper (Woźniak et al., 2017a). Besides, as mentioned above, O2•− content increased in the roots and in leaves in response to higher Pb dose. High O2•−content was maintained after 24 h. However, at 24 h time-point, O2•− levels decreased in the roots but increased in the leaves as compared with 0 h. This observation may be explained by the fact that Pb at higher concentration is transferred from roots to leaves, which is a known mechanism increasing tolerance to Pb. As reported by Fahr et al. (2013), Pb affects plants primarily through their root system that rapidly responds by synthesis and deposition of callose that forms a barrier preventing Pb entrance, by the uptake of large amounts of this element and its sequestration in the vacuole, or by its translocation to the aboveground parts. Also, in 24-h roots of pea seedlings infested by aphids and growing at high Pb concentration, we observed higher generation of O2•− than in the non-infested seedlings. At the same time-point, O2•− level in the presence of high Pb dose was lower in the leaves infested by aphids than in the non-infested ones. Lower content of O2•− in 24-h leaves of 0.5 mM Pb(NO3)2+aphids variant than in 24-h leaves of 0.5 mMPb(NO3)2 variant may be associated with rapid dismutation of this ROS to H2O2; the level of H2O2 was the highest in 24-h leaves. Application of the hormetic Pb dose did not cause such an increase in O2•− production in the organs of pea seedlings as the sublethal Pb dose. The accumulation of O2•− in the leaves of pea seedlings infested by aphids and growing in the presence of the hormetic dose of Pb was noted at 24 hpi but it was lower than in the leaves exposed to the sublethal dose. Then, the accumulation of this ROS as a result of a low concentration of Pb and A. pisum infestation was observed at 72 hpi. Increase in the levels of O2•− in Pb treated pea seedlings and its quick dismutation to H2O2 with the participation of

sublethal concentration for pea seedlings may have a deterrent effect on A. pisum and thus reduce their feeding. It may be related to the fact that Pb reduces plant water potential and cell turgor (Sharma and Dubey, 2005). Aphids feeding on pea seedlings exposed to the sublethal Pb dose (0.5 mM Pb(NO3)2) showed a trend towards increasing the total duration of the xylem phase. This may indicate the necessity to supplement the phloem sap with water, the activity observed when aphids were deprived of primary symbionts and in need to regulate osmotic potential (Table 2) (Pompon et al., 2011). The results of assayed oxidative stress indexes also suggest that Pb may influence aphids indirectly through ROS activity. Our findings revealed that the levels of O2•− increased considerably in roots and leaves of pea seedlings, particularly those growing at the sublethal dose of Pb as well as during cross-interactions of both stress factors, i.e. Pb and A. pisum (Fig. 2). In roots, O2%− levels remained high at all time points of the experiment (Fig. 2A). More intense O2%− generation in the roots (Fig. 2A) than in the leaves (Fig. 2B) may result from the direct contact of the roots with this heavy metal. One of the causes for excessive production of this ROS in pea seedling organs may be reduced SOD activity (Fig. 5A, B). Thus we conclude that overproduction of this ROS may constitute an important line of defence of pea seedlings against A. pisum. SOD zymogram showed that Mn-SOD was hardly detected in the roots of seedlings growing at the sublethal dose of Pb and during Pb and aphid interaction. It may be assumed that the activity of Mn-SOD in these tissues may be limited by low level of Mn2+ recorded in our experiments in pea seedling organs detected by EPR (Fig. 4c and d). Despite low SOD level in the stressed seedling organs, accumulation of O2•− in the roots was several times greater than in the leaves (Fig. 2). As already stated above, this may result from the direct contact of roots with Pb. On the other hand, this finding may also suggest that SOD efficiency in the leaves of seedlings grown in the presence of the sublethal Pb dose and 14

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number of phloem phases and the number of sustained sap ingestion phases. In the same experimental model (Woźniak et al., 2017a), administration of Pb at the sublethal concentration (0.075 mMPb(NO3)2) triggered abundant accumulation of this element in the roots in comparison with the leaves. Pb administered at the hermetic dose was mainly accumulated in the roots, while only small amounts appeared in the leaves. Lower content of Pb in the leaves than in the roots can actually indicate the existence of barriers limiting the transport of this metal from the roots to the above-ground parts of plants. One of these barriers is endoderm (e.g. Casparian strips), which acts as a physical boundary (Pourrut et al., 2011). This barrier, however, is not always fully effective, as demonstrated by the example of terrestrial and aquatic plants (Zea mays, Seregin et al., 2004, Lemna minor, Kocjan et al., 1996). Kafel et al. (2010) showed that plants from polluted areas accumulated higher concentrations of Pb and Zn in the leaves and shoots, while Cd was more abundant in the shoots. The shoots and leaves of plants infested with aphids contained higher Zn but lower Pb levels. Research literature comprises reports stating that herbivorous insects feeding on contaminated plants may suffer from developmental disorders manifested for example by an alteration of some life history traits, such as changes in morphology. Cabbage aphids (Brevicoryne brassicae L.) reared on Pb contaminated plants were smaller and showed a considerable degree of morphological asymmetry when compared with aphids feeding on non-contaminated plants (Görür, 2006a, 2006b). Results of spectrophotometric analyses also showed that generation of H2O2 in the leaves of pea seedlings exposed to the low dose of Pb at the beginning of the experiment (i.e. four days after the administration of Pb) was much higher than in the control seedlings (Fig. 3b), while the generation of O2•− and semiquinone radicals in these leaves was lower or only slightly higher than in the control, respectively (Fig. 2b). We assume that high level of H2O2 in the leaves of seedlings treated with the low dose of Pb may be connected with the signalling role of that molecule. Additionally, as shown previously in the leaves of pea seedlings from 0.075 mM Pb(NO3)2 variant at 0 h, an increase in the activity of phenylalanine ammonialyase (PAL) and accumulation of total salicylic acid (TSA) were much greater than in the control leaves (Woźniak et al., 2017a). Also, high level of hydrogen peroxide, considerably higher than in the control, was observed at 0 h in the leaves of pea seedlings treated with the sublethal dose of Pb (Fig. 3). At the next time point, i.e. at 24 h after infestation, a very strong increase was observed in the generation of H2O2 in the affected leaves (+aphids variant) and in the leaves treated with high level of Pb and infested by A. pisum (0.5 mM Pb(NO3)2+aphids variant) (Fig. 3b). The increase in H2O2 levels at 24 hpi resulted in enhanced activity of peroxidase towards the phenolic substrate, i.e. syringaldazine (POX), at the subsequent time point, i.e. 48 hpi. Hydrogen peroxide as POX substrate may be used in the formation of lignins, and the presence of these compounds may hinder aphid feeding. A reduction in the number of the phloem phases and number of sustained sap ingestion phases at high concentration of Pb was confirmed in the EPG recordings. As reported by Morkunas and Gmerek (2007), POX involved in lignification shows high affinity to its substrate syringaldazine (Imberty et al., 1985), especially its anion forms located in the cell wall (Boudet, 2000; Marañón and Van Huystee, 1994). Our experiments demonstrated that POX capable of participating in tissue lignification was strongly stimulated in the organs of pea seedlings both by Pb presence and feeding of A. pisum. Moreover, spectrophotometric analyses showed that transferring aphids onto pea seedlings treated with the hormetic dose of Pb did not trigger H2O2 accumulation in the leaves (0.075 mM Pb(NO3)2+aphids variant). An interesting finding is connected with enhanced generation of H2O2 in 72-h roots of the seedlings exposed to 0.075 mM PbNO3 and infested by pea aphids (0.075 mM Pb(NO3)2 +aphids) vs. the variant of 0.075 mM Pb(NO3)2. An increase in the level of this molecule in the above-mentioned roots indicates systemic signal transmission from

SOD justifies the choice of this ROS as an indicator of the oxidative stress. Moreover, the low level of Mn2+ detected by EPR spectroscopy particularly in the tissues exposed to the toxic dose of Pb, may be connected with limited uptake of these ions from the medium as a result of antagonism between Pb2+ and Mn2+ (Christensen et al., 1979; Sharma and Dubey, 2005). The drop in Mn2+ content was observed in the roots but not in leaves. As reported by Hansel et al. (2012), a decrease in Mn2+ may be due to formation of Fe/Mn plaques on root surface and to increased sequestration of Pb. Similar reduction in the activity of SOD upon Pb exposure was observed in humans (Patil et al., 2006). Perhaps this Pb effect observed in our study originates from an impairment in photosynthetic electron transport chain, and does not relate directly and specifically to the defence mechanism against the heavy metal or aphids infestation. Photosystem I (PSI) is the main O2•− and consequently H2O2 producer in chloroplasts (Romanowska et al., 2008). However, SOD activity was higher in the roots than in the leaves. In this paper, we also demonstrate that lipid peroxidation indicates oxidative stress in cells. The levels of thiobarbituric acid reactive substances (TBARS) at the sublethal doses of Pb in the organs of P. sativum seedlings both infested and non-infested by A. pisum were higher than in the control and other variants. TBARS content in the leaves was higher than in the roots. This may be associated with higher activity of the antioxidant enzymes, e.g. SOD activity was higher in the roots than in the leaves. Dias et al. (2019) reported on higher Pb accumulation, oxidative damage and changes in phytohormone pools in the roots than in the leaves. EPR spectrometry showed relatively stable levels of semiquinone free radicals in pea seedlings (Fig. 4a and b). These organic radicals are formed via addition of a single hydrogen atom with its electron to a quinone or via removal of a single hydrogen atom with its electron from the corresponding hydroquinone. Semiquinone radicals show high reactivity and cytotoxicity, as reported by Hammerschmidt (2005). Together with O2•− they may be incorporated into polymers such as lignins and thus may protect the cell wall against degradation and limit cell penetration by insects such as aphids (Mai et al., 2013). Results of EPR experiments indicate that the concentration of semiquinone radicals, similarly as of O2•−, in the roots of pea seedlings exposed to the sublethal Pb dose and the combined interaction of Pb and A. pisum was considerably higher than in the other experimental variants. This result suggests that the heavy metal is the stressor inducing generation of semiquinone radicals in roots, while feeding of pea aphids additionally enhances its generation, possibly via a systemic signal transfer from the leaves to the roots. In the leaves of seedlings growing in the presence of the toxic dose of Pb, generation of these organic radicals generally shows a different trend than in the other experimental variants. Their highest level was recorded in the leaves exposed to low concentration of Pb and infested by A. pisum (0.075 mM Pb(NO3)2+aphids variant) and in the leaves infested by the aphids alone (+aphids variant). A key factor of these responses seems to be connected with the aphid feeding that causes mechanical injury and affects the plants through elicitors found in aphid saliva. Our inference was confirmed by the EPG results, in which the total duration of non-probing before the first phloem phase was the shortest among the tested variants. As it was even shorter than in the control seedlings, it may indicate a stimulation of piercing, accompanying wounding. The cumulative percentage of aphids on pea seedlings growing at the low dose of Pb (0.075 mM Pb(NO3)2+aphids variant), indicating a higher number of phloem phases, was greater than in the control plants (Table 2, Fig. 8). We observed a slightly positive effect of Pb at the low concentration (0.075 mM Pb(NO3)2 variant) on aphid behaviour in comparison with the control and seedlings exposed to 0.5 mM Pb(NO3)2. The onset of the first phloem phase was significantly accelerated and the duration of an individual period of sap ingestion was slightly longer in 0.075 mM Pb(NO3)2 seedlings. Also, the total duration of sap ingestion was the longest and the non-probing time was the shortest in 0.075 Pb(NO3)2 seedlings. In contrary to its low concentration, Pb at the high concentration seemed to reduce the 15

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5. Conclusions

leaves to roots (Capone et al., 2004) as a result of frequent piercings related to probing by A. pisum. As mentioned previously, the time of probing until the phloem was reached was 1.5 times shorter than in the control (35.99 min vs. 53.25 min) (Table 2). The confocal microscope images demonstrated that relative generation of H2O2 in the leaf epidermal layer was the strongest at 24 h after feeding by pea aphids and in the variant with toxic Pb dose (Fig. 3c). At the subsequent time point (48 hpi), the strongest green fluorescence was observed in the cells of the leaf epidermal layer in the seedlings exposed to the hormetic dose of Pb and infested by A. pisum (0.075 mM Pb(NO3)2+aphids). The fluorescence was also visible at 72 hpi (Fig. 3c). The strongest fluorescence indicating the presence of H2O2 was observed primarily in the cell wall region, while a weaker signal was recorded in the cytoplasm. H2O2 was visible in the guard cells in all experimental variants but the strongest fluorescence was observed in the leaf cells of pea seedlings infested by aphids. In the context of the systemic response, a particularly interesting finding is connected with a considerable effect of A. pisum feeding on the level of H2O2 in 72 h roots of pea seedlings cultured with low level of Pb and infested by A. pisum (0.075 mM Pb(NO3)2+aphids variant) (Fig. 3a). This result may indicate systemic signal transmission from leaves to roots. Literature sources indicate generation of H2O2 and O2•− in plants responding to aphid feeding (Borowiak-Sobkowiak et al., 2016; Czerniewicz et al., 2017). For example, H2O2 observed specifically at the site of aphid feeding (de Ilarduya et al., 2003) was also implicated to take part in aphid defence (Kuśnierczyk et al., 2008). A mutation in Arabidopsis respiratory burst oxidase homolog D (RbohD) gene, resulting in decreased H2O2 accumulation increased the plant sensitivity to aphids (Miller et al., 2009). Moreover, in our experiments, stereomicroscopy images revealed increased fluorescence indicating H2O2 generation in the surface layer of the aphid body after 72 h feeding on pea leaves cultured in the presence of Pb. Stronger fluorescence emission was observed in aphids feeding on seedlings treated with Pb than in the control aphids (Fig. 3d). Moreover, fluorescence emission in aphids feeding on pea seedlings exposed to the toxic dose of Pb was higher than in aphids feeding on the seedlings treated with low level of Pb. As mentioned above, aphids on the pea seedlings treated with the toxic Pb dose accumulated much more Pb in their bodies than those feeding on seedlings growing in the presence of low Pb dose (Woźniak et al., 2017a). This finding supports our earlier discussion concerning the effect of Pb alone as a stress factor on A. pisum. Moreover, the effect of ROS alone or cytotoxic semiquinone radicals generated by pea seedlings on A. pisum may not be excluded. Evidence for H2O2/ROS production in phytophagous insects such as Aphididae is well-documented (Madhusudhan and Miles, 1998; Miles, 1999; Urbańska, 2008). As reported by Madhusudhan and Miles (1998), H2O2 is produced when phenolics are oxidized by saliva of A. pisum (Harris) and Therioaphistrifolii maculate (Buckton). Also Urbańska (2009) provided some data on the identification and production of H2O2 in Rhopalosiphumpadi and Sitobionavenae, in particular on H2O2 synthesis in the digestive system and salivary secretions. Therefore, we should not exclude the possibility that H2O2 detected in pea seedling tissues after aphid feeding may be to some degree of the aphid origin. Various Pb concentrations in the substrate affected total antioxidant capacity (TAC) (Fig. 6a, d). The high dose of Pb enhanced TAC dependent on the pool of slow- (residues of tyrosine and tryptophan in proteins) and fast-acting (ascorbic acid or glutathione) antioxidants in the roots and leaves infested and non-infested by A. pisum. An opposite trend was observed for TAC dependent on the pool of slow- and fast antioxidants, in the roots and leaves growing at the toxic Pb dose both infested and non-infested by A. pisum (Fig. 6b, d). In conclusion, pea seedlings both infested and non-infested by A. pisum showed positive and negative correlations between increased/decreased antioxidant activity and the toxic dose of Pb. Additionally, our previous studies revealed that Pb and aphid attack synergistically affected SA and pisatin accumulation (Woźniak et al., 2017a).

The results of this study indicate that the response of pea seedlings to the heavy metal and aphids differed greatly at low and toxic Pb concentration. The intensity of these stress oxidative defence responses and antioxidant system depended on the organ, the metal dose and direct contact of the stress factor with the organ. The results of our analyses presented in this study will improve understanding of the plant–aphid interactions under various heavy metal contamination levels and the role of these insects in the trophic chain, thus providing novel information on plant and aphid biology.The study also revealed that in pea seedlings growing at low concentration of Pb, A. pisum tends to reduce its non-probing activity before the first phloem phase, reach phloem phase sooner, and increase the duration of sap ingestion as compared with control plants. Additionally, low concentration of Pb does not reduce aphid longevity but, contrary to the toxic does, its lightly reduces their net rate of reproduction. Author contributions AW, PhD student of IM designed the study, wrote and prepared the manuscript, performed most of the experiments, and analyzed and interpreted the data; IM created the concept and designed the study (this concept was the basis of a research project for the Polish National Science Centre-NCN, grant number 2017/25/N/NZ9/00704), analyzed literature and wrote the manuscript, analyzed and interpreted the data, supervised the organization of the study; WB contributed to the measurements of semiquinone radicals and manganese ion concentrations using the electron paramagnetic resonance (EPR) technique; KD supervised the EPG experiments and contributed to the analysis of EPG data; BG, designed the EPG experiments and interpreted the EPG results; BB-S contributed to the analysis regarding determination of the effect of Pb on the demographic parameters of A. pisum population; JB contributed to elementary comparisons between particular levels of the analyzed factors at different time points using the two-sample t-test for equal means for all observed traits; SS contributed to the analysis of images using a confocal microscope; RR-S contributed in detection of SOD isoforms, analyzed and interpreted the data and discussion section of the manuscript Funding This work was supported by the National Science Centre, Poland, grant number 2017/25/N/NZ9/00704. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jplph.2019.152996. References Abrahams, P.W., 2002. Soils: their implications to human health. Sci. Total Environ. 291, 1–32. Ai, T.N., Naing, A.H., Yun, B.-W., Lim, S.H., Kim, C.K., 2018. Overexpression of RsMYB1 enhances anthocyanin accumulation and heavy metal stress tolerance in transgenic petunia. Front. Plant Sci. 9. https://doi.org/10.3389/fpls.2018.01388. Anjum, S.A., Ashraf, U., Khan, I., Tanveer, M., Ali, M., Hussain, I., Wang, L.C., 2016a. Chromium and aluminum phytotoxicity in maize: morpho-physiological responses and metal uptake. Clean – Soil Air Water 44, 1075–1084. https://doi.org/10.1002/ clen.201500532. Anjum, S.A., Ashraf, U., Khan, I., Tanveer, M., Saleem, M.F., Wang, L., 2016b. Aluminum and chromium toxicity in maize: implications for agronomic attributes, net photosynthesis, physio-biochemical oscillations, and metal accumulation in different plant parts. Water Air Soil Pollut. 227, 326. https://doi.org/10.1007/s11270-016-3013-x. Asada, K., Yoshikawa, K., Takahashi, M., Maeda, Y., Enmanji, K., 1975. Superoxide dismutases from a blue-green alga, Plectonema boryanum. J. Biol. Chem. 250, 2801–2807. Ashraf, U., Kanu, A.S., Deng, Q., Mo, Z., Pan, S., Tian, H., Tang, X., 2017. Lead (Pb) toxicity; physio-biochemical mechanisms, grain yield, quality, and Pb distribution

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