Plant Science 221–222 (2014) 1–12

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Differential induction of Pisum sativum defense signaling molecules in response to pea aphid infestation ˙ e, Van Chung Mai a,b , Kinga Drzewiecka c , Henryk Jelen´ d , Dorota Narozna f g ´ ˛ Renata Rucinska-Sobkowiak , Jacek Kesy , Jolanta Floryszak-Wieczorek a , h a,∗ Beata Gabry´s , Iwona Morkunas a

Department of Plant Physiology, Pozna´ n University of Life Sciences, Woły´ nska 35, 60-637 Pozna´ n, Poland Department of Plant Physiology, Vinh University, Le Duan 182, Vinh City, Viet Nam c Department of Chemistry, Pozna´ n University of Life Sciences, Wojska Polskiego 75, 60-625 Pozna´ n, Poland d Institute of Plant Products Technology, Pozna´ n University of Life Sciences, Wojska Polskiego 31, 60-624 Pozna´ n, Poland e Department of Biochemistry and Biotechnology, Pozna´ n University of Life Sciences, Dojazd 11, 60-632 Pozna´ n, Poland f Department of Ecophysiology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 60-614 Pozna´ n, Poland g Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Gagarina 9, 87-100 Toru´ n, Poland h Department of Botany and Ecology, University of Zielona Góra, Prof. Szafrana 1, 65-516 Zielona Góra, Poland b

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Article history: Received 22 September 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 31 January 2014 Keywords: Pisum sativum Acyrthosiphon pisum Jasmonic acid Salicylic acid Ethylene Nitric oxide

a b s t r a c t This study demonstrates the sequence of enhanced generation of signal molecules such as phytohormones, i.e. jasmonic acid (JA), ethylene (ET), salicylic acid (SA), and a relatively stable free radical, nitric oxide (NO), in response of Pisum sativum L. cv. Cysterski seedling leaves to the infestation of pea aphid Acyrthosiphon pisum (Harris) at a varied population size. In time from 0 to 96 h after A. pisum infestation these signal molecules accumulated transiently. Moreover, the convergence of these signaling pathways occurred. JA and its methyl derivative MeJA reached the first maximum of generation at 24th hour of infestation. An increase in ET and NO generation was observed at 48th hour of infestation. The increase in SA, JA/MeJA and ET concentrations in aphid-infested leaves occurred from the 72nd to 96th hour. In parallel, an increase was demonstrated for the activities of enzymes engaged in the biosynthesis of SA, such as phenylalanine ammonia-lyase (PAL) and benzoic acid 2-hydroxylase (BA2H). Additionally, a considerable post-infestation accumulation of transcripts for PAL was observed. An increase in the activity of lipoxygenase (LOX), an important enzyme in the biosynthesis of JA was noted. This complex signaling network may contribute to the coordinated regulation of gene expression leading to specific defence responses. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Aphids feed on the phloem sap of the host plant using stylets of their sucking–piercing mouthparts [1]. During penetration, aphid stylets pierce through the epidermis, mesophyll, as well as other parenchymatous tissues until they reach the phloem. The stylets penetrate tissues mainly through the apoplast, although frequent

Abbreviations: ACC synthase, 1-aminocyclopropane-carboxylate synthase; ACC oxidase, 1-aminocyclopropane-carboxylate oxidase; BA2H, benzoic acid 2hydroxylase; cADPR, cyclic ADP ribose; ET, ethylene; FW, fresh weight; GACC, 1-(␥glutamyl)-ACC; hpi, hours post-infestation; JA, jasmonic acid; JA/MeJA, jasmonates; LOX, lipoxygenase; MACC, 1-(malonyl)-ACC; MTA, 5 -methylthioadenosine; NO, nitric oxide; PAL, phenylalanine ammonia-lyase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SA, salicylic acid; SAM, S-adenosyl-l-methionine. ∗ Corresponding author. Tel.: +48 61 8466040; fax: +48 61 8487179. E-mail addresses: [email protected], [email protected] (I. Morkunas). 0168-9452/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plantsci.2014.01.011

punctures of the symplast are also observed [2]. The factors responsible for the activation of plant defence comprise not only mechanical damage, but also elicitors which are present in aphid saliva [3]. Salivation is crucial for successful colonization by aphids, and saliva is thought to play a major role in aphid virulence [4]. Evidence suggests that aphids, like other plant parasites, deliver repertoires of proteins inside their hosts that function as effectors to modulate host cell processes. These insects most likely secrete effectors into their saliva while progressing through the different plant cell layers during probing and feeding [5]. Functional analyses showed that one of these proteins, Mp10, induced chlorosis and weak cell death, and suppressed the oxidative burst induced by the bacterial PAMP flg22. The recognition of aphid infestation by plants likely occurs through the use of transmembrane pattern recognition receptors (PRRs) or, acting largely inside the cell, polymorphic nucleotidebinding site-leucine-rich repeat (NBS-LRR) protein products, encoded by a majority of resistance genes [6]. This activation

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may induce defensive reactions which are the result of highly coordinated sequential changes at the cellular level comprising, among other changes, the synthesis of signal molecules. Following the recognition of an attacker, plants use different signaling cascades to reprogram their phenotype. Typical signal molecules in the plant defence mechanisms include phytohormones such as jasmonic acid (JA), salicylic acid (SA), ethylene (ET), and reactive oxygen/nitrogen species (ROS/RNS), mainly hydrogen peroxide (H2 O2 ), and nitric oxide (NO), which induce alteration in the expression of defence genes, leading to specific metabolic changes enhancing plant defence responses [7]. These molecules can act separately or together, with antagonistic or synergistic interactions in the plant signaling network. Our previous studies demonstrated a strong generation of H2 O2 as a signaling molecule triggering defence mechanisms in leaves of Pisum sativum L. cv. Cysterski seedlings in response to infestation of pea aphid Acyrthosiphon pisum (Harris) at a varied population size [8]. Accumulating evidence suggests that ROS signaling pathways are closely interwoven with hormone-signaling pathways in plant–insect interactions [9]. A different role of JA in plant–aphid interactions has been described in several plants such as Arabidopsis, barrel clover, tobacco, tomato, wheat and sorghum [7]. Beside JA, its precursor, 12-oxo-phytodienoic acid (OPDA), and JA methyl ester (MeJA) are also essential elements in plant defence mechanism [10]. Analysis of microarray data suggested the defensive function for JA signaling as JA-inducible defence genes are transiently induced in both compatible and incompatible tomato-aphid interactions [11]. Several genes encoding enzymes required for JA synthesis and JA-mediated defence responses, such as 12-oxophytodienoate 10,11-reductase, cytochrome P450, were up-regulated in aphid-resistant plants after the attack of aphids [12,13]. Some isozymes of lipoxygenase (LOX), the key enzyme in the JA biosynthesis pathway, were also induced in plant defence response to aphid infestation [14,15]. Furthermore, JA is known as a signal molecule, an enhanced generation of which occurs in plants after wounding by herbivores [16]. In turn, only a few available reports demonstrated the role of SA in plant defence response to aphid infestation and its expression was varied in different plant species. The accumulation of SA related to aphid feeding in cereals provided evidence that indicated a possible involvement of this phytohormone in plant defence mechanism [17–19]. At the transcriptional level, a number of SA-defenserelated genes have been demonstrated to be related to the defence response of several plant species. Transcript abundance of genes associated with the SA signaling pathway, including pathogenesisrelated (PR) genes such as ˇ-glucanase 2, chitinases increased in sorghum to defend against greenbug, Schizaphis graminum (Rondani) [15]. The action of the non-expressor of the PR1 (NPR1) gene was necessary for Arabidopsis to induce PR gene responses to the green peach aphid, Myzus persicae (Sulzer) [14]. Expression of gene encoding phenylalanine ammonia-lyase (PAL – an enzyme involved in the biosynthesis of SA) and other SA-responsive transcripts such as PR5, PR10 and ˇ-glucanase were activated by infestation of the bluegreen aphid, Acyrthosiphon kondoi Shinji, in both resistant and susceptible variants of legume Medicago, although there were some differences in the magnitude and kinetics of the induction [20]. On the other hand, strong generation of ET was observed in susceptible cultivars of alfalfa and wheat early after infestation by S. graminum and the spotted alfalfa aphid, Therioaphis maculata (Buckton) [21,22]. In turn, ET accumulation in barley was associated with Diuraphis noxia (Kurdjumov) susceptibility, but was positively correlated with resistance to Rhopalosphum padi (L.) and S. graminum [23,24]. Interestingly, the burst of ET detected in tomato after Macrosiphum euphorbiae (Thomas) feeding was similar to that in plant–pathogen and other plant–herbivore interactions [25]. The expression of genes encoding proteins involved in ET production

or ET signaling (e.g. sterol -7 reductase, ethylene-responsive elements) was up-regulated in aphid-susceptible celery infested with M. persicae [26] and in aphid-resistant wheat infested with D. noxia [13]. ET production was enhanced in both resistant and susceptible plants in response to aphids, suggesting that ET may be involved in basal defence against these phloem-feeding insects. Recent advanced research revealed the cross-talk of JA, SA, and ET in a complex network of interconnecting signaling pathways, which allows plants to develop the optimum defensive strategies. SA and JA are known to interact antagonistically in plant responses to herbivore attacks, and SA often suppresses endogenous production of JA when it reaches a certain level [27]. The synergistic interaction between SA and JA, however, has been described in reaction of several plants to piercing-sucking herbivores. Infestation of D. noxia elicited the SA- and JA/ET-dependent signaling pathways in wheat by mimicking aspects of both pathogen and herbivorous insect attacks [28]. The simultaneously increased SA and OPDA accumulation caused by the Hessian fly Mayetiola destructor (Say) larvae was a possible result of a coordinated interaction between SA and JA pathways in regulating wheat resistance to this type of insect [19]. Rather than being the principal elicitor of herbivore-induced responses, ET plays a more subtle role in modulating other defense signals [29]. Regardless of the sequence of activation, ET and JA appear to interact synergistically in a majority of defence responses. For example, resistance to M. persicae in Arabidopsis was related to an increasing level of ET and the expression of the ethylene insensitive 2 (EIN2) gene – a bifunctional transducer of ET and JA signal transduction [30]. Data suggest that JA, ET, and SA signaling may be involved in certain forms of innate resistance of plants to aphids. Thompson and Goggin [31] reported that innate resistance in plant–aphid systems is due to differences in the timing and magnitude of plant defences, rather than to qualitatively different responses. The initial recognition and signaling events are likely to be rapid, fleeting, and highly localized to the feeding site. MeJA/ET-responsive basic ␤-1,3-glucanase in tomato showed only a weak, local and transient accumulation in response to aphids, and patterns of induction did not differ between compatible and incompatible interactions [11]. The signaling pathways are driven not only by phytohormones, but also by ROS/RNS, including hydrogen peroxide and nitric oxide that ultimately contribute to the production of plant defense proteins and/or secondary compounds [32]. Nitric oxide (NO), which is the subject of research in this study, has been demonstrated to be an essential regulator of several physiological processes in plants [33]. NO can exert its biological function through different ways, such as the modulation of gene expression, the mobilization of secondary messengers (changes in cytosolic Ca2+ concentrations, the activation of cGMP synthesis), or interplays with various protein kinases [34,35]. Beside these signaling events, NO can be responsible for the posttranslational modifications (PTM) of target proteins [33,36]. Moloi and van der Westhuizen [37] demonstrated that in two Triticum aestivum L. varieties, D. noxia feeding induced early production of NO to higher levels in resistant plants than in susceptible ones. On the other hand, nitric oxide (NO) is known also to be involved in many physiological reactions of insects. For example, Ganassi et al. [38] suggest that NO, prevalently synthesized by the calcium/calmodulin-dependent isoform of nitric oxide synthase (NOS), plays important physiological roles both in adult and embryological stages of aphids. The first aim of this study was to verify whether the enhanced generation of signal molecules such as JA, ET, SA and NO occurs in the leaves of P. sativum L. cv. Cysterski seedlings in response to the pea aphid infestation at a varied population size. The study included the estimation of the intensity and time scale of signaling molecule generation. The sequence of generation of these signal molecules in defence response of pea seedling leaves to the pea aphid in time

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from 0 to 96 h was thus determined. The second objective was to determine the activity of enzymes engaged in the biosynthesis of the listed signal molecules, such as phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) and benzoic acid 2-hydroxylase (BA2H) in the SA synthesis pathway and lipoxygenase (LOX, EC 1.13.11.12) – an important enzyme in the synthesis of JA. Next to LOX activity, changes in the isoenzymatic spectrum of this enzyme were also determined. In the available literature, the information regarding the sequence of enhanced generation of signal molecules in plant response to aphid infestation is still missing. It should be stressed that the aforementioned aspects of the research as well as the monitoring of NO generation in leaves of P. sativum seedlings after infestation by A. pisum are novel problems. 2. Material and methods 2.1. Material Experiments in this study were carried out on seedlings of edible pea (P. sativum L. cv. Cysterski), whose seeds were obtained from the Plant Breeding Company at Tulce near Poznan (Poland). Seeds were surface-sterilized, immersed in sterile water and left in an incubator at 23 ◦ C. After 6 h of imbibition, the seeds were transferred onto filter paper in Petri dishes and immersed in a small amount of water in order to support further absorption. After a subsequent 18 h, the imbibed seeds were sown in 20-cm-diameter pots containing sterilized perlite. Pea seedlings were kept in a growth chamber at 22–23 ◦ C, 65% relative humidity, and light intensity of 130–150 ␮M photons m−2 s−1 with the 14/10 h (light/dark) photoperiod. The objects of this study were leaves of pea seedlings. The following variants were applied: (1) control pea seedlings, with no infestation by pea aphids (A. pisum (Harris)); (2) pea seedlings infested by A. pisum populations of various sizes, i.e. 10, 20 and 30 aphids per plant. Leaves of pea seedlings were carefully collected at 0, 24, 48, 72 and 96 h of infestation (hpi) by aphids. Material was weighed, and then was immediately frozen in liquid nitrogen for subsequent analyses of JA/MeJA and SA as well as LOX, BA2H, and PAL activity. Detection of ET production and monitoring of relative NO generation were performed in fresh materials at particular time points for all variants. 2.2. Aphids and infestation experiment The aphid species used was the pea aphid (A. pisum (Harris)), originally cultured and supplied by the Department of Entomology (Poznan´ University of Life Sciences, Poland). It was reared on its host, P. sativum L., in the growth chamber at a temperature of 22–23 ◦ C, humidity of 65%, photoperiod of 14/10 h (light/dark) with light intensity of 130–150 ␮M photons m−2 s−1 . On the 10th day of culture in perlite each pea seedling was infested with 10, 20, or 30 apterous adult females of A. pisum. In the experiments 10day-old seedlings were used. This developmental stage proved to be the most suitable for aphids, which was demonstrated by high daily fecundity (preliminary experiments). Pea aphid development is dependent on the age of the plant; generally, developmentally younger plants with high turgor are better infested by the pea aphid than developmentally mature plants. Aphid specimens were carefully transferred to leaves with a fine paintbrush. The aphid populations were monitored through all experiments. The number of apterous females of A. pisum remained constant in each experiment, because the newly born larvae were removed two times a day. The control plants in experiments were pea seedlings with no pea aphids. All control and aphid-infested seedlings were put separately in glass boxes (30 cm × 28 cm × 30 cm) covered by nylon

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gauze, and placed in a growth chamber with the environmental factors controlled. 2.3. Determination of jasmonates The analyses of jasmonates, including JA and MeJA, were carried out following the minor modification from GC–MS method described by [39]. The pea leaves were shock-frozen in liquid nitrogen and stored at −80 ◦ C until extraction. Material (1.0 g) was homogenized with 200 ng of deuterium-labeled methyl jasmonate (d2-MeJA) and 200 ng of deuterium-labeled jasmonic acid (d5-JA) as internal standards in 20 mL of 80% (v/v) methanol. The suspension was left to stir overnight and then centrifuged at 10,000 × g for 10 min. The residue was re-extracted with 80% methanol in two parts of 20 mL each. Combined supernatants were reduced to the aqueous phase by rotary evaporation, acidified to pH 2.0 with 7 M HCl and centrifuged at 10,000 × g for 10 min to remove chlorophyll. The supernatant was partitioned three times against chloroform, then dried under vacuum. The residue was dissolved in 3 mL n-hexane and applied to a silica gel solid-phase extraction column (Backer-bound SPE silica gel, 500 mg, 3 mL; J.T. Backer, Philipsburg, NJ, USA). The column was washed with 5 mL n-hexane and then eluted with 6 mL of n-hexane:diethyl ether (2:1, v/v) with 0.5% (v/v) acetic acid. The eluate was evaporated and further purified by HPLC using a SUPELCOSIL ABZ + PLUS column (250 mm × 4.5 mm, 5 ␮m particle size; Supelco Inc., USA). The samples were dissolved in 200 ␮L of 20% methanol and chromatographed with a linear gradient of 20–80% methanol in 1% (v/v) formic acid in 20 min, flow rate 1.0 mL/min at temperature 22 ◦ C. The fractions collected at the retention times of 12.0–13.4 min for JA and 18.30–19.20 min for MeJA were evaporated to dryness, in the case of JA methylated with (Trimethylsilyl)diazomethane (Sigma–Aldrich), dissolved in 50 ␮L of methanol and analyzed by GC–MS-SIM (Auto-System XL coupled to a Turbo Mass, Perkin Elmer) using a DB-5 column (30 m × 0.25 mm, 0.5 ␮m phase thickness). The GC temperature program was 80 ◦ C for 1 min, 80–160 ◦ C at 10 ◦ C/min, 160–230 ◦ C at 5 ◦ C/min, flow rate 1 mL/min, injection port 250 ◦ C, electron potential 70 eV. GC/MS-SIM analysis was performed by monitoring m/z 156, 161, 193, 198, 224, 226 and 229. The dwell time for all ions was 100 ms. Level of JA and MeJA in the samples were determined from the ratio of peak area, calculated following d5-JA and d2-MeJA standards, and expressed as nanograms per gram of fresh material (ng g−1 FW). 2.4. Determination of salicylic acid Salicylic acid in the free form (SA) was extracted and quantified following the HPLC method as described [40] with minor modifications. Frozen leaves were ground in liquid nitrogen to a fine powder, from which approximately 0.50 g was taken for analysis. SA was extracted twice with methanol 90%, strongly stirred and centrifuged again at 12,000 × g for 10 min at 4 ◦ C. After centrifugation, the supernatant was divided into two equal parts and the solvent was evaporated to dryness under a stream of nitrogen. Each part was extracted three times with the extractive organic mixtures of ethyl acetate:cyclopentane:isopropanol (100:99:1, v/v/v). After solvent evaporation, the dry residue was dissolved in a mobile phase (200 mM acetate buffer, pH 5.0 and 0.5 mM EDTA) and analyzed by HPLC coupled with fluorometric detection in a WATERS Company chromatograph (Milford, MA, USA) composed of a 2699 Separation Module Alliance and 2475 Multi-␭ Fluorescence Detector. Chromatographic separation was run on a Spherisorb ODS2 WATERS Company column (3 ␮m, 4.6 mm × 10 mm). Detection parameters were 295 nm for excitation and 405 nm for emission.

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The content of SA was calculated and expressed as nanograms per gram of fresh weight material (ng g−1 FW).

␮M trans-cinnamic acid performed per mg protein per hour (␮M trans-cinnamic acid mg−1 protein h−1 ).

2.5. Determination of ethylene

2.9. Total RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis

Measurements of ET generation in pea seedling leaves were performed by the GC method. An Agilent 7890A GC instrument with an S/SL injector and a flame ionization detector (FID) was used with a GsBP-PLOT Al2 O3 “KCl” GC column (50 m × 0.32 mm × 8.0 ␮m). Fresh leaves from the control and aphid-infested pea plants were placed in 20 mL glass vials sealed with a Teflon/silicon crimp cap. The volume of 1.0 mL of air from the vials was injected with a gastight syringe into the GC instrument with the isothermal temperature of 110 ◦ C, injector temperature of 120 ◦ C and FID at 240 ◦ C with helium as a carrier gas flowing at 1.0 mL/min. The amount of ET was quantified by comparing the peak height of sample peaks with the standard curve using custom prepared ET in nitrogen of 0.318 ± 0.006 (v/v; Multax, Poland), and was expressed in nanoliters per one gram of the fresh sample (nL g−1 FW). 2.6. Determination of LOX isoenzymes The spectrum of LOX isoenzymes was analyzed following the isoenzymatic method as described [41]. Leaves were homogenized in 0.2 M borate buffer (pH 7.0) and centrifuged at 13,000 × g for 15 min. Proteins isolated with the borate buffer were electrophoresed in 7% polyacrylamide gel containing ampholytes (pH 3.5–10.0). Isoenzymes were visualized by staining with 1% N,Ndimethyl-p-phenylene diamine.

Pea seedling leaves (0.50 g) were frozen in liquid nitrogen, and ground with a mortar and pestle in the presence of liquid nitrogen. For RT-PCR analyses of a target gene, total RNA was isolated from 45 mg tissue using the SV Total RNA Isolation System (Promega) according to the recommendations of the manufacturer [44]. This protocol was designed to process small tissue samples. The RNA level in samples was spectrophotometrically assayed at 260 nm. The A260 /A280 ratio varied from 1.8 to 2.0. RT-PCR was performed in the GenAmp PCR System 9700 (Applied Biosystems). Total RNA was used in the first-strand synthesis using Verte M-MLV reverse transcriptase (Novazym) in a 25 ␮L reaction mixture with oligodT primers according to the manufacturer’s protocol. A volume of 1 ␮L of the first-strand reaction mixture was then PCR-amplified for 30 cycles using Allegro Tag Polymerase DNA (Novazym) in 25 ␮L reaction mixture according to the manufacturer’s protocol. Optimization of PCR reaction conditions (temperature and time of the individual steps, the number of cycles, the concentration of DNA polymerase, the concentration of Mg2+ and primers) was performed. The PCR products were then analyzed on agarose gel. The sequences of the specific primers for PAL were used for PCR reactions, such as the forward primer for PAL: 5 -CCAAGTCAATTGAGAGGGAG-3 the reverse primer for PAL: 5 -CATCTTGGTTGTGCTGCTC-3

The following actin primers were used as positive controls: the forward primer for actin: 5 -GCATTGTTGGTCCTCCTCG-3 the reverse primer for actin: 5 -TGTGCCTCATCCCCAACATA-3

2.7. Lipoxygenase assay The enzyme LOX (EC 1.13.11.12) was extracted and measured following the spectrophotometric method [42]. Frozen leaves (0.50 g) were ground with a mortar and pestle, adding 3 mL 100 mM phosphate buffer (pH 7.0). The homogenate was then centrifuged at 12,000 × g for 30 min at 4 ◦ C. The supernatant was directly used as a crude extract for LOX activity assay. The linoleic acid solution was prepared at a concentration of 10 mM. The substrate solution consisted of 100 mM borate buffer (pH 10.0), Tween 20, and 10 mM linoleic acid. The reaction mixture consisted of 2.9 mL borate buffer, 50 ␮L substrate, and 50 ␮L enzymatic extract. The increase in absorbance was monitored at 234 nm wavelengths. An absorbance increase of 0.001 was taken as one unit (U) of LOX activity. The activity of the enzyme was expressed as U per 1 mg protein per minute (U mg−1 protein min−1 ). 2.8. Phenylalanine ammonia-lyase assay The activity of PAL (EC 4.3.1.24) was determined with a modified method of Cahill and McComb [43]. The amount of 0.50 g of frozen leaves was homogenized at 4 ◦ C with a mortar and pestle in 4 mL of 100 mM Tris–HCl buffer (pH 8.9) containing 5 mM ␤-mercaptoethanol, and 0.050 g PVP. After that, the homogenate was centrifuged at 12,000 × g for 20 min at 4 ◦ C. The supernatant was used for enzyme analysis. The reaction mixtures contained 0.50 mL of 20 mM borate buffer (pH 8.9), 0.50 mL of 10 mM lphenylalanine, and 0.50 mL extract in a total volume of 1.5 mL. A sample without the substrate l-phenylalanine was used as a blank. The reaction proceeded for 24 h at 30 ◦ C and was interrupted by the addition of 1.5 mL of 2 N HCl. PAL activity was measured by the change of absorbance at 290 nm due to the formation of trans-cinnamic acid using a Perkin Elmer Lambda 15 UV-VIS spectrophotometer (Norwalk, CT). The activity of PAL is expressed as

2.10. Benzoic acid 2-hydroxylase assay BA2H, an enzyme catalyzing the hydroxylation of benzoic acid to form SA, was measured using HPLC according to [45] with minor modifications. The amount of 0.50 g of frozen leaves was ground in a chilled mortar, and the resulting fine powder was suspended in 2 mL of extractive buffer consisting of 20 mM HEPES (Sigma–Aldrich, pH 7.0), 12.5 mM 2-mercaptoethanol, 10 mM sorbitol, 1% PVP (w/v), and 0.1 mM PMSF. The suspension was vortexed and then centrifuged at 12,000 × g for 15 min. All extraction procedures were carried out at 4 ◦ C. The supernatant was used for enzyme assays. In a final volume of 0.50 mL the reaction mixture contained of 20 mM HEPES buffer (pH 7.0), 1 ␮M of BA, 1 ␮M of NAD(P)H (Sigma–Aldrich), extractive buffer and enzyme extract in equal volume. The BA2H reaction mixture was incubated for 30 min at 30 ◦ C in the water bath, and thereafter, 250 ␮L of 15% TCA was added to stop the reaction. Then after vortexing and centrifuging at 12,000 × g for 10 min, SA obtained as the product of reaction catalyzed by BA2H was extracted from the supernatant three times with 0.50 mL of the extractive organic mixtures of ethyl acetate:cyclopentane:isopropanol (100:99:1, v/v/v). Each time the organic layer was collected to obtain the total volume of 1.50 mL and then the solvent was evaporated to dryness. The sample was dissolved in 250 ␮L mobile phase immediately before use. The entire mixture was thoroughly mixed, then transferred to Ependorf tubes and centrifuged at 15,000 × g for 2 min. After centrifugation, the volume of 200 ␮L was transferred into the HPLC vial fitted with a rubberized Teflon-sept and the contribution of PP and placed in the autosampler chamber. The concentration of salicylic acid (ng ␮L−1 ) was assessed. The injection volume was constant (10 ␮L) and the same for samples and standards. SA concentration was analyzed by the HPLC method coupled with fluorometric detection using a WATERS Company

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chromatograph (Milford, MA, USA) composed of a 2699 Separation Module Alliance and 2475 Multi-␭ Fluorescence Detector. Chromatographic separation was run on a Spherisorb ODS2 WATERS Company column (3 ␮m, 4.6 mm × 10 mm). Detection parameters were 295 nm for excitation and 405 nm for emission. Activity of BA2H was expressed as the number of nanograms of SA obtained as a reaction’s product during 1 h for the enzyme extracted from 1 mg of protein (ng SA h−1 mg−1 protein). Protein content in pea seedling leaves was determined according to Bradford [46] using bovine serum albumin (Sigma–Aldrich) as a standard. 2.11. Monitoring of nitric oxide generation Relative generation of nitric oxide was detected using a fluorescent dye, 4,5-diamino fluorescein diacetate (DAF-2DA, Calbiochem) [47] with some modification. Fresh pea leaves were placed and incubated for 12 h with 20 mL of 100 ␮M DAF-2DA in loading buffer (10 mM Tris–HCl, pH 7.2) in 3 mM dimethylsulphoxide (DMSO) in darkness at room temperature. The leaf sections were then washed three times with fresh loading buffer to remove excess fluorophore and were observed under a confocal microscope (the model Zeiss LSM 510, Axioverd 200 M, Jena, Germany with a filter set No. 10, excitation 488 nm, emission 520–530 nm or more). The fluorescence of chloroplasts was captured with a 585 nm long-pass filter or larger. Images were obtained at the same depth and were analyzed by the Image Browser software (version 4.2).

Fig. 1. The effect of Acyrthosiphon pisum infestation on the level of jasmonates (JA and MeJA) in seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05).

which was 1.74-fold higher than in the control. ANOVA results showed that the differences in JA/MeJA concentration in aphidinfested variants and the control plants at 24 hpi and 96 hpi were highly statistically significant (e.g., P = 0.00008 and P = 0.00203 at infestation by 30 aphids, respectively).

2.12. Statistical analysis All determinations were performed in three independent experiments. Analysis of variance (ANOVA) was applied to verify whether means from independent experiments within a given experimental variant were significant. Data shown in the figures are means of triplicates for each variant and standard errors of mean (SE). In individual figures significant differences are shown using asterisks. Statistical analysis was based on Student’s t-test with the level of significance ˛ = 0.05.

3.2. Accumulation of SA in pea seedling leaves after pea aphid infestation

3.1. Accumulation of JA in pea seedling leaves after pea aphid infestation

Infestation of A. pisum induced the accumulation of SA in P. sativum L. cv. Cysterski seedling leaves. In aphid-infested leaves, SA accumulated to higher levels than the control at 48 h, and increase more dramatically at 96 hpi (Fig. 2). The highest SA concentration was 290.33 ng g−1 FW in leaves infested by 20 aphids at 96 hpi, which was by 8.85-fold higher than at the beginning of the experiment and by 5.72-fold higher than in control plants. Although there was no positive correlation in accumulated SA content with the density of pea aphid infestation, significant differences between SA levels in aphid-infested leaves and the control were recorded from 48 hpi to 96 hpi.

GC/MS-selected ions of JA and MeJA in the extract of P. sativum L. cv. Cysterski leaves were performed by monitoring m/z 156, 161, 193, 198, 224, 226 and 229. The dwell time for all ions was 100 ms. Calculated intensity of investigated ions showed that leaves of pea seedlings mainly contained JA, whereas MeJA was present in very small amounts. Total jasmonate content refers to levels of JA and MeJA determined jointly in all samples; however, abbreviation JA was used as representative. Endogenous levels of JA/MeJA in aphidinfested leaves were generally significantly different from that in control plants (Fig. 1). A high infestation rate (30 aphids) results in a rapid increase in JA/MeJA, followed by a drop then a second pulse, whereas infestation with fewer aphids (10, 20) results in a delayed accumulation of jasmonates starting at 48 h. The content of this signal molecule changed twice during infestation time. At the first enhancement at 24 hpi, the levels of jasmonates were proportional to the numbers of pea aphids on plants, and the highest concentration of JA/MeJA (443.17 ng g−1 FW) was recorded in leaves infested by 30 aphids, which was 1.80-fold and 3.24-fold higher than at the beginning of the experiment and in comparison with the control, respectively. At the second increase, the greatest induction of JA/MeJA was caused by the infestation of 20 aphids/plant, as the highest content of jasmonates at 96 hpi was 484.87 ng g−1 FW,

Fig. 2. The effect of Acyrthosiphon pisum infestation on the level of SA in seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05).

3. Results

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Fig. 3. The effect of Acyrthosiphon pisum infestation on the level of ET released from seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05). Fig. 4. The effect of Acyrthosiphon pisum infestation on LOX activity in seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05).

3.3. Accumulation of ET in pea seedlings after pea aphid infestation Generation of ET in aphid-infested leaves was at statistically higher levels than in the control during the experiment and it remarkably increased as soon as the infestation of pea aphids started up to 48 h (Fig. 3). The amount of ET generated at 48 hpi was proportional to the pea aphid population sizes. The highest ET production was recorded in leaves infested by 30 aphids (49.1 nL g−1 FW), which was by 3.27- and 3.10-fold higher than that at the beginning of infestation and in the control, respectively. ANOVA results showed that the differences in ET concentration in aphid-infested variants and the control plants were highly statistically significant (e.g., P = 0.00066 at infestation by 30 aphids at 48 hpi). Following the reduction of ET at 72 hpi, there was a second enhancement of ET that reached a high level at 96 hpi (P = 0.0059; infestation by 30 aphids). In contrast, in control plants a small amount of ET (12.5–15.8 nL g−1 FW) was produced and it changed little from the beginning up to 96 hpi.

3.4. LOX activity and isoenzymes spectrum in pea seedling leaves after pea aphid infestation Activity of LOX increased continuously from the beginning of the experiment up to 96 hpi (Fig. 4). Significant differences in LOX activity were observed in the period from 24 to 96 h between the control and aphid-infested variants as shown in Fig. 4. Although LOX activity was not related to the impact of different numbers of A. pisum per plant, the evidence that this enzyme in aphid-infested leaves expressed significantly higher activity than in the control at all time-points of the experiment shows that the described increase in its activity could have been caused by aphid infestation. Moreover, results of electrophoretic analyses revealed the occurrence of two LOX isoforms in pea seedling leaves (Fig. S1, supplementary data). Infestation of A. pisum caused an enhancement in the expression of these isoforms. The first isoform was expressed at high level in 7% IEF slab gel from the beginning up to 48 hpi, whereas staining intensity of the second isoform was stronger than the first one starting from 72 hpi. Expression of both two LOX isoforms in aphid-infested variants was higher than in the control. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.plantsci. 2014.01.011.

3.5. Activity and gene expression level of PAL in pea seedling leaves after pea aphid infestation There was a higher level of PAL activity in pea seedling leaves infested by pea aphids when compared with the control plants, and the alteration in PAL activity was proportional to the population sizes of pea aphid and the duration of infestation (Fig. 5A). Activity of PAL in aphid-infested leaves increased from the beginning up to 96 h of the infestation experiments. The enzyme activity in 20- and 30-aphid-infested leaves showed great enhancement starting from 72 hpi, it reached the highest values at 96 hpi, which was 4.51-fold and 4.69-fold higher than at the beginning (0 h), and 5.5-fold and 5.2-fold than in the control, respectively. In the control pea seedling leaves, the activity of this enzyme remained at a low level and changed little throughout the experiment. Significant differences in PAL activity were observed at 72 and 96 h of aphid infestation among the experimental variants as analyzed by ANOVA. A considerable post-infestation accumulation was observed using semi-quantitative RT-PCR for PAL transcripts. Relative transcript levels of phenylalanine ammonialyase (PAL) genes began to express in A. pisum-infested leaves since 24 hpi. However, the expression of PAL was strongly enhanced at 72 hpi and 96 h (Fig. 5B). Relative transcript levels of PAL genes were generally higher in aphidinfested leaves than in the controls.

3.6. Activity of BA2H in pea seedling leaves after pea aphid infestation The BA2H activity in leaves of pea seedlings changed little from the beginning up to 48 hpi in all variants (Fig. 6). The difference in activity of this enzyme between the control and aphid-infested plants was recorded from 72 hpi. The activity of BA2H in the control leaves slightly increased from the beginning up to 72 h of culture and then decreased, while in aphid-infested leaves this activity increased remarkably. The highest BA2H activity (52.25 ng SA h−1 mg−1 protein) was detected at 96 h after infestation by 20 aphids, which was 3.28-fold higher than at the beginning, and 3.15-fold higher in comparison to the control plants. Significant differences in BA2H activity were observed among the experimental variants as analyzed by ANOVA.

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examined under a confocal laser microscope. Fluorescent DAF-DA penetrates living cells, where it is hydrolyzed by cytosolic esterase to release DAF-2, which in the presence of NO is converted to the fluorescent triazole derivative (DAF-2T). The DAF-2DA staining fluorescence (green color) appeared in the P. sativum leaves infested by A. pisum (Fig. 7). The strongest staining and the largest area of fluorescence on the surface of aphid-infested leaves were observed at 48 hpi, then the intensity of fluorescent dye was quenched from 72 up to 96 hpi. A small difference between the fluorescence intensities in leaves infested by different numbers of pea aphids was recorded at each time point of the measurement time. In the control non-infested leaves, NO-dependent green fluorescence was low, probably due to constitutive NO production. In turn, the infestation of A. pisum induced NO accumulation as the defense response of P. sativum. This green fluorescence signal was expressed at the highest levels at 48 hpi. However, relative NO generation in pea seedling leaves was not proportional to population sizes of pea aphids.

4. Discussion

Fig. 5. (A) The effect of Acyrthosiphon pisum infestation on PAL activity in seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05). (B) RTPCR products amplified from the PAL gene derived from pea seedling leaves of the control and infested by A. pisum (a), constitutive expression of actin (b) and a ratio of intensity of PAL/intensity of actin (c).

3.7. Monitoring of nitric oxide generation Relative generation of nitric oxide in pea seedling leaves was investigated by staining leaves with the specific indicator DAF-2DA

Fig. 6. The effect of Acyrthosiphon pisum infestation on BA2H activity in seedling leaves of Pisum sativum L. cv. Cysterski. Significance between control and aphid infestations was determined by ANOVA and is represented by * (P < 0.05).

Numerous plant biochemical pathways regulating plant defense, development and metabolism are simultaneously or sequentially involved in these interactions [48]. In response to attack by insects, including aphids, plants activate signaling cascades, leading to the accumulation of signal molecules such as endogenous hormones and relatively stable free radicals that trigger the induction of defense. Signaling pathways are driven by phytohormones such as JA, ET, SA, abscisic acid (ABA), giberellic acid, and by free radicals such as H2 O2 and NO [7]. In the present study we showed how the pea aphid (A. pisum) infestation influences the intensity and the duration of generation of the analyzed signal molecules in the leaves of P. sativum L. cv. Cysterski seedlings. Moreover, we discuss the roles of these molecules in plant responses to the phloem-feeding aphids. The results of this study revealed the sequence of the postinfestation enhanced generation of JA/MeJA, ET, SA and NO in leaves of pea seedlings (Fig. 8). Previous studies showed an enhanced post-infestation H2 O2 generation in leaves of P. sativum L. cv. Cysterski seedlings [8]. In the period from 0 to 96 h after A. pisum infestation all studied signal molecules were accumulated transiently. Moreover, the convergence of these signaling pathways was also observed. At certain time points of aphid infestation the relationship between aphid population size and the intensity of generated signal molecules was demonstrated (Figs. 1 and 3). The activity of enzymes engaged in the biosynthesis of the above mentioned signal molecules, such as LOX in the JA biosynthesis pathway (Fig. 4) and PAL (Fig. 5A and B) and BA2H in the SA biosynthesis pathway (Fig. 6), was observed to increase. Next to the increase in LOX activity (Fig. 4), the occurrence of two LOX isoforms in pea leaves was also revealed (Fig. S1) and the expression of these isoforms was enhanced after A. pisum infestation. It has been revealed that following aphid infestation these signal molecules were induced at different points of time. A strong generation of JA/MeJA and H2 O2 [8] was observed in pea seedling leaves at 24 h of infestation (Figs. 1 and 8). After the induction of ET and NO at 48 h of infestation in aphid-infested leaves (Figs. 3 and 7), the generation of SA increased strongly at subsequent time points (72 and 96 hpi) (Fig. 5). Subsequently, JA (Fig. 1) and ET (Fig. 3) were again induced and reached the second maximum levels at 96 h of infestation. Therefore, at 96 h there was a combined action of three phytohormones, i.e. SA, JA and ET in the defence mechanism of pea leaves (Fig. 8). The important role of JA and its derivatives in response to aphid feeding has been previously shown in various plants [49]. In pea

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Fig. 7. Relative generation and cytochemical localization of nitric oxide in seedling leaves of Pisum sativum L. cv. Cysterski in the control and infested by Acyrthosiphon pisum population of various sizes, e.g. 10, 20, 30 aphids per plant in the period from 0 to 96 h of culture. Green fluorescence came from DAF-2DA (diaminofluorescein-2 diacetate), which was observed under a Zeiss LSM 510 confocal microscope (objective magnification 20×); scale bar 50 ␮m.

seedling leaves, JA/MeJA accumulation (Fig. 1) and the increase in the activity of the enzyme LOX in the JA-biosynthesis pathway (Fig. 4) resulted from pea aphid infestation. As the earliest inducible phytohormone, the enhancement of JA/MeJA at 24 hpi (Fig. 1) may be related to the functioning of JA as a signal molecule that triggers defense mechanisms in pea leaves against aphids. Additionally, the first LOX isoform in the period from 24 to 48 hpi was enhanced and its expression was much stronger than in the control (Fig. S1). The high expression of the second LOX isoform from 72 to 96 hpi was recorded and its intensity increased with time after infestation and was much stronger than in the control. One can assume that probably the first isoform was involved in the enhancement of generation of JA in pea seedling leaves in the period from 24 to

48 hpi, while for the second isoform it was in the period from 72 to 96 hpi. JA is known primarily as a signal in wound response [50]. Although aphids minimize tissue damage during feeding, probing does result in cell wall disturbance, disruption of plasma membranes, and penetration of epidermal, mesophyll, and parenchyma cells; the degree of injury that occurs during probing varies considerably among aphid species [2,4]. There was some evidence of wound-specific JA responses in plants after aphids attack, and patterns of induction did not differ between compatible and incompatible interactions [11,15]. More recent research, however, recorded activation of JA-mediated defense upon attack by aphids, which try to limit tissue damage during feeding [15,20,51–53].

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Fig. 8. The sequence of signal molecules such as phytohormones, i.e. jasmonic acid (JA), ethylene (ET), salicylic acid (SA), and a relatively stable free radical, nitric oxide (NO), enhanced in responses of Pisum sativum L. cv. Cysterski seedling leaves to the infestation of pea aphid Acyrthosiphon pisum (Harris) at a varied population size. The figure shows the intensity and time scale of the generation of phytohormones (JA, ET, SA) and free radical NO. Additionally the figure includes hydrogen peroxide (H2 O2 ), which relative generation and cytochemical localization in P. sativum L. cv. Cysterski seedling leaves infested by A. pisum was shown in time from 0 to 96 h of culture [8]. The diagram presents general trends for the level of signaling molecule generation in pea seedling leaves depending on the infestation of A. pisum at 10, 20, 30 aphids per plant.

In addition, JA mediates systemic wound responses through distinct cell autonomous and non-autonomous pathways [54]. In both pathways bioactive JAs are recognized by the receptor system that couples hormone binding to ubiquitin-dependent degradation of transcriptional repressor proteins. On the other hand, JA signaling functioned as the inducible herbivore defense. The induction of JA responses in some aphid-infested plants was weaker than in mechanically wounded plants. As an important enzyme in the JA-biosynthesis pathway, LOX showed a defensive role in some aspects. The involvement of LOX in JA synthesis that regulates plant responses to wounding was recorded in various plants such as Arabidopsis, cabbage, tobacco and tomato [55]. LOX was also a part in the inducible herbivore resistance, which has been implicated in coordination of direct plant defence and indirect plant defence through genotypic and phenotypic induction and inhibition [56]. Genetic interference in LOX production resulted in reduced volatile emission, defence gene expression, attraction of parasitoids and increased plant damage. Evidence showing a strong increase in the levels of transcripts encoding LOX after aphid infestation has been mentioned in the defence response of Arabidopsis, tobacco, wheat and sorghum [12–14,51]. However, LOX genes and other genes involved in JA biosynthesis were only marginally and transiently induced by aphids [15,20]. LOX in pea seedling leaves (Figs. 4 and S1) may not only play an important role in defence response, but it can also control some other processes, because its increase was observed in the course of time, both in the leaves of the control and in aphid-infested leaves. For example, LOX has been associated with some processes in a number of developmental stages [57]. LOX is also an important storage protein during vegetative growth [58]. The increased SA level may be a critical step in the signaling of down-stream defence responses in plants to aphid infestation. Total SA began differential accumulation to higher levels in resistant rather than susceptible wheat within 24 h after aphid infestation [18]. The SA concentration in resistant plants increased with time and reached a peak at 96 hpi, whereas, in the susceptible

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plants SA accumulation was much less pronounced and was most noticeable after 48 h of infestation. Our results are largely consistent with these reports because infestation with pea aphids resulted in dramatic increases in SA accumulation up to 96 hpi in the infested leaves of cv. Cysterski (Fig. 2). Expression of some SA-defence-related genes was induced in the plant defence against aphids [14]. The NPR1 protein is a vital component of SA signaling in Arabidopsis, because NPR1 supplies an important link between different defense mechanisms, for example, the action of the NPR1 gene was necessary for Arabidopsis to induce PR gene responses to aphids, or the enhanced PDF1.2 induction in the npr1 mutant. Along with the involvement of defence genes, several SA-responsive transcripts in the legume Medicago truncatula Gaertn., such as BGL, PR5, and PR10, were activated by A. kondoi infestation in both resistant and susceptible plants, although there were some differences in the magnitude and kinetics of the induction [20]. A wide range of defence responses of Arabidopsis to attack by Brevicoryne brassicae (L.), depending on SA-signaling, was involved in the induction of the following transcription factors of the WRKY family [52]. Other genes involved in SA synthesis (ICS1 and ICS2), induction of the SA-signaling pathway (EDS1, EDS5 and PAD4) and stress-responsive SA-dependent genes (PR1, PR2, and PR4) were also up-regulated. Because infestation activities induce defence reactions in plants, the increase in SA levels in pea seedlings suggests that this phytohormone might take part in the signaling of downstream responses to pea aphids. PAL is a key enzyme of the phenylpropanoid pathway, where SA can be synthesized. Activity of PAL and the expression of the gene encoding this enzyme in P. sativum seedling leaves were induced after A. pisum infestation. Similar results were found in other plants following aphid attack [11,12,14,15,20]. The increase in PAL activity and expression of the PAL gene (Fig. 5A and B) suggested that pea seedling leaves can strongly synthesize secondary metabolites, including SA, related to anti-insect capability. However, PAL is the starting point for a whole range of metabolites that are induced upon aphid infestation and therefore it is difficult to state with any significant degree certainty that PAL induction is specifically associated with SA accumulation. In turn, BA2H, a soluble cytochrome P-450 monooxygenase, which major biochemical function is to catalyze the conversion of benzoic acid directly to SA, is directly related to SA biosynthesis. Early studies reported the function of BA2H in plant defence against pathogens [40,43]. However, there is a lack of information on the involvement of BA2H in plant response to herbivores, including aphids. Our results revealed that infestation by A. pisum caused an increase in BA2H activity in P. sativum seedling leaves (Fig. 6). The inducible change in the activity of this enzyme was closely related with the alteration of the SA level, similarly as PAL activity. As the first result presented in the plant–insect interactions, we showed BA2H participation in SA biosynthesis in infested pea seedling leaves. Aphids are known to promote an increase in ET emission in several plant–aphid interactions. For example, in barley the ET level was strongly increased in the D. noxia-susceptible variety, but it was low in the aphid-resistant variety [23]. Both the susceptible and resistant varieties produced ET during infestation by another aphid, S. graminum. Maximum production of ET occurred 48 h after infestation by both D. noxia and S. graminum and then declined to baseline levels. However, in other susceptible varieties of barley, maximum ET production was detected after 16 h, when plants were infested with 10 nymphs of S. graminum [24]. In the aphid pea infested cultivar ‘Cysterski’ the enhanced production of ET in aphid-infested leaves to a high level was observed at 48 hpi and ET content was clearly dependent upon the number of aphids per plant, reaching a maximum when plants were infested with 30 pea aphids (Fig. 3). Furthermore, ET in pea seedling leaves expressed

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the second induced increase at 96 hpi, while the highest ET level was observed in 20-aphid-infested leaves. Plants modulate ET content in response to aphid infestation at the level of synthesis, transport, by regulating perception and signal transduction, and/or expression of genes. ET induces the synthesis of compounds that cross-link with cell wall polymers, thus reinforcing the cell wall structure and reducing injury caused by feeding activities [24]. In the plant defence mechanism ET has been shown to elicit some defensive proteins/enzymes, such as polyphenoloxidase and peroxidase, that are thought to form quinones, which inhibit digestion in the insect gut [29]. When ET was first mentioned as a possible signal mediating defence response in tomato, accumulation of the proteinase inhibitor 2 transcript was shown to depend on ET and JA synergistically [59]. In addition, ET signaling has been implicated in the activation of the accumulation of nicotine, phenolics, aliphatic glucosinolate, terpenoid resin and sesquiterpene, which all functioned as other defence responses in plants [29]. One of the primary molecular effects of ET was to alter the expression of various target genes. Generally, genes involved in the ET pathway, e.g. ACO (1-aminocyclopropane-1-carboxylate oxidase), ERF1 (ethylene responsive factor), HEL (hevein-like protein) were induced in both the susceptible and resistant plants of M. truncatula following A. kondoi infestation [20]. In addition, ET increased the transcript levels of defence-related genes, including PRs, chitinase, ˇ-1,3 glucanase, peroxidase, chalcone synthase, as well as cysteine proteinase genes [60]. However, ET does not regulate the expression of PR genes alone, but rather in a complex interaction of signaling molecules [61]. In the plant signaling network ET was known to elicit an enhanced production of ROS in response of some plants. For example, ET emission significantly increased after aphid infestation in defence of barley against S. graminum and R. padi. At the same time, H2 O2 accumulation and soluble peroxidase activity increased in barley infested with 20 aphids of S. graminum. The aphids induced an increase in H2 O2 accumulation with a transient maximum after 20 min of infestation and it was 41% higher than in non-infested plants [24]. However, the emission of ET in P. sativum leaves occurred later than H2 O2 burst; the high content of ET was firstly noted at 48 hpi and then observed at 96 hpi. We previously reported that H2 O2 generation rapidly increased, reaching a maximum at 24 h after infestation [8]. In this study we revealed that at 24 h after infestation JA and its methyl derivative MeJA also reached the first maximum of generation. Defence mechanisms underlying the cross-talk between JA and ET signaling pathways are poorly understood [7]. ET is required for the concomitant induction of JA or other signals by modulating the sensitivity to the second signals [62]. It has been demonstrated in legume M. truncatula that ET seems to contribute to the herbivory-induced terpenoid biosynthesis by modulating both the early signaling events such as cytoplasmic Ca2+ -influx and the downstream JA-dependent biosynthesis. JA and ET-dependent pathways have frequently been demonstrated to synergistically or antagonistically induce plant defence responses to herbivores. Generally, the ET signaling pathway exhibited a synergistic interaction with JA signaling via production of proteins such as proteinase inhibitors, defensins [63]. However, in some cases aphid feeding increased ET levels and the expression of several genes essential for the ET signaling pathway was found to be in a negative relation with JA [30]. Results of our study indicate that the simultaneous enhanced generation of certain hormones or free radicals is found at the same time points, which may suggest their synergistic action. Therefore, in the leaves of pea cv. Cysterski seedlings under aphid infestation JA and ET may be assumed to interact antagonistically or synergistically, depending on the time of induction. The negative interaction was expressed in the first response. JA accumulation reached a high

level at 24 hpi, whereas ET was not elicited. When ET emission reached a high level at 48 hpi, JA content was reduced. The positive relation was found in the later responses of pea, as the second induction of both JA and ET was recorded at 96 hpi. JA and SA are known to interact antagonistically in plant responses to herbivores, and when it reaches a certain level SA often suppresses endogenous production of JA [27]. Suppression of effective JA defences by the induction of the less efficient SA signaling-based responses appeared in cabbage when B. brassicae manipulated plant-induced resistance [51]. Similarly, other aphids such as S. graminum, M. euphorbiae and M. persicae strongly induced up-regulation of the SA-dependent pathway and reduced the expression of JA-dependent genes. Apparently, aphids inhibit an efficient plant defence conferred by JA-regulated genes, while allowing the SA-regulated pathway to propagate [64]. In those cases the time of JA and SA accumulation is often different. Our study revealed that a high JA level perhaps suppress endogenous production of SA in the first response of pea seedling leaves at 24 hpi; JA/MeJA was strongly accumulated, whereas SA accumulation was maintained at a low level. In turn, in the later responses of pea (72–96 hpi), the contents of the two phytohormones were remarkably high. Infestation of aphids elicited the JA- and SA-signaling pathways by mimicking aspects of both pathogen and herbivorous insect attacks [28]. Taken together, data presented here suggest that phytohormones JA, SA, and ET constituted important lines in the defense responses of P. sativum to A. pisum infestation. Following aphid infestation, JA, SA, and ET were accumulated in leaf cells at different time points. JA was first induced and reached high levels at 24 hpi as the inducible herbivore defence and/or wounding specific reaction. ET emission at 48 hpi may lead to the activation of other responses involved in the expression of numerous defensive proteins and defence-related genes. As the finally accumulated phytohormone at 96 hpi, SA might take part in the signaling of downstream responses of pea to pea aphid infestation. In addition to SA, the second enhancement of JA and ET suggested a positive interaction of these phytohormones in the later response of pea. Moreover, the enzyme LOX involved in JA biosynthesis, as well as PAL and BA2H in the SA biosynthesis pathway were also elicited by infestation of pea aphids and were correlated with the enhanced accumulation of JA and SA, respectively. The induction of these enzymes contributed to the strengthening of the phytohormonal barriers in pea defence against pea aphid infestation. Goodspeed et al. reported that jasmonates are required for enhanced herbivory resistance when entrained in-phase with Trichoplusia ni loopers, and jasmonates and salicylate accumulation patterns show circadian rhythms with an opposite phasing. These results suggest the possibility that the clock-controlled jasmonate fluctuations lead to the circadian accumulation of defense metabolites. Moreover, NO was also involved in the wounding response to phloem-feeding insects such as the brown planthopper (BPH) [65]. Within minutes of mechanical wounding, a strong burst of NO release was triggered [66]. Similarly, an NO burst was observed within 1 h of infestation with BPH in both resistant and susceptible genotypes [65]. In our study infestation of A. pisum induced NO accumulation in the defence response of P. sativum and this signal molecule demonstrated the highest generation at 48 hpi (Fig. 8). During plant responses to aphids NO seems to act upstream of SA or interact with the superoxide anion (O2 ·− ) to form peroxynitrite (ONOO− ) [37]. In addition, a local and rapid accumulation of NO in wounded epidermal cell layers of Arabidopsis leaves revealed NO as an early player of response to stress [66]. The potential role of NO as a modulator of plant wounding responses has been addressed through its cross-talk with other signal molecules such as JA, H2 O2 [66,67].

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SA, JA, and ET are plant-specific hormones involved in the response to the attack by many aphids in a broad range of plant species. JA, ET and SA signaling cascades do not activate defences independently, but activate complex interactions. The interactions between SA, JA and ET defense signaling pathways are dependent on the plant species, the organisms attacking the plants, and the developmental phase and physiological state of the plant. In conclusion, in the presented study we identified unique differences in responses associated with the generation of signaling molecules by pea cv. Cysterski in response to A. pisum infestation. Study of the time course of defense signaling molecules production upon pea aphid (A. pisum) colonization, measurements of phytohormone levels, relative NO generation, quantification of induced PAL and LOX enzymatic activity and observations of the growth of seedlings of P. sativum cv. Cysterski may indicate that it is a tolerant cultivar of pea. As reported by [68] one of the forms of aphid resistance is tolerance where the plant is able to grow despite aphid infestation. Results of research presented in this study will contribute to a better understanding of regulation mechanisms during the plant–aphid interactions, thus providing novel information to our knowledge on plant biology.

Acknowledgement The study was supported by the Polish National Science Centre (NCN, Grant No. 2011/01/B/NZ9/00074).

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