Tetraneura ulmi (Hemiptera: Eriosomatinae) Induces Oxidative Stress and Alters Antioxidant Enzyme Activities in Elm Leaves

Tetraneura ulmi (Hemiptera: Eriosomatinae) Induces Oxidative Stress and Alters Antioxidant Enzyme... Abstract Gall formation is induced by an insect, which changes normal plant development and results in the formation of a new organ, following distinct stages of metabolic and developmental alterations. Research on mechanisms of recognition and responses to biotic stress may help to understand the interactions between galling aphids and their host plants. In this study, Tetraneura ulmi L. (Hemiptera: Eriosomatinae) galls and Ulmus pumila L. (Rosales: Ulmaceae) leaves were used as a model. Concentrations of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances, electrolyte leakage, as well as the activity of ascorbate peroxidase, guaiacol peroxidase, and catalase (CAT) were determined in galls and two parts of galled leaves (with and without visible damage). Biochemical analyses were performed at three stages of gall development: initial, fully developed, and mature galls. A slight increment in H2O2 content with a strong enhancement of ascorbate peroxidase and CAT activities were observed in galls and galled leaves in the first stage. In subsequent stages of gall development, a progressing increase in H2O2 production and cell membrane damage was associated with declining antioxidant enzyme activities, especially in gall tissues. The stages of gall development are likely to be part of cell death triggered by aphid feeding. It seems that the gall is the result of a biochemical struggle between the host plant and the gall inducer. aphid, biotic stress, gall development, plant defense Most insect herbivores have very intimate associations with their host plants. Galling aphids are of special interest, because they change normal plant development by stimulating tissue differentiation and new organ formation (Wool 2004). The activity of galling herbivores causes structural and biochemical alterations in host plant tissues. Initiation, increment in mass and qualitative differentiation, maturation, and senescence are key phases of gall development (Raman 2011). The isolation and insulation of one or a few cells on the plant from their normal course of differentiation is the earliest event in gall induction. These cells are irreversibly changed by an external stimulus from the inducing insect (Raman 2012). Aphid galls are formed only on young, growing plant tissues, and their colonization activity is timed with the host plant phenology (Wool 2004, Oliveira et al. 2016). The Tetraneura ulmi L. aphid forms bean-shaped galls on young leaves of different elm species (Ulmus sp.) in the spring (Kmieć and Kot 2007, Blackman and Eastop 2018). Each gall is induced by a single female (fundatrix) emerging from fertilized egg. Aphid propagates in the gall by parthenogenetic reproduction. Wingless progeny becomes winged in June and disperses from the cracking gall to find the secondary host. Only two generations of aphids develop in the gall (Urban 2003, Kmieć and Kot 2007). Aphids use their slender and flexible stylets to penetrate plant tissue intercellularly to access the sieve elements for feeding (Walling 2008, Giordanengo et al. 2010). However, occasionally, stylets also puncture cells and minute amounts of cell contents are ingested (Tjallingii 2006). During feeding, insects introduce effectors that alter plant defense signaling, infestation symptoms, and plant development (Giordanengo et al. 2010, Rodriguez and Bos 2013). However, no cecidogenic compounds were identified in aphid saliva; hypertrophy-inducing triacylglycerides were isolated from Colopha moriokaensis (Monz.) body (Ohta et al. 2000). Although aphids feed primarily from the phloem, the identified effectors could also mediate plant–aphid interactions in other cell types or in intercellular spaces (Will et al. 2013). Plants have the ability to respond to insect infestation by changing their biochemical states. Early defense responses to insect feeding include protein phosphorylation, membrane depolarization, calcium influx, and release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2; Maffei et al. 2007, Giordanengo et al. 2010). A low level of ROS is maintained by the presence of various secondary plant metabolites and scavenging enzymes under normal plant developmental conditions (Gill and Tuteja 2010). According to Giordanengo et al. (2010) and Mai et al. (2013), aphid feeding induces oxidative burst and an increase in ROS production in the host plant. Despite their potential toxicity, ROS in fact play a dual role in vivo (Sharma et al. 2012). Lower doses of ROS are employed as signals that mediate at least part of the stress responses (Hung et al. 2005), while at higher concentrations, they are part of the direct defense and pose a significant threat that may eventually lead to programed cell death (PCD) (Sharma et al. 2012). ROS can provoke reversible or irreversible modifications of proteins, causing alterations in the regulation of plant metabolism and activation of transcriptional processes (Gill and Tuteja 2010). H2O2 is a strongly depolarizing molecule among ROS that can be induced by the feeding insect. H2O2 levels increase in response to herbivores as long as the attacks persist (Maffei 2007). An increase in ROS production is accompanied by higher lipid peroxidation under stress conditions. Degradation of polyunsaturated fatty acids can lead to chain breakage, thereby increasing membrane fluidity and permeability (Sharma et al. 2012, Bhattacharjee 2014). Efficient enzymatic and nonenzymatic defense plant system are activated to cope with the elevated ROS levels (Maffei et al. 2007). Peroxidases and catalase (CAT) are among the enzymes that play an important role in plant stress caused by insect feeding. The presence of oxidative enzymes at H2O2 generation sites can serve to detoxify ROS that accumulate in response to abiotic and biotic stresses (War et al. 2012). Ascorbate peroxidase (APX, EC 1.1.11.1) is regarded as one of the most widely distributed antioxidant enzymes in plant cells, and APX isoforms have significantly higher affinity for H2O2 than CAT, making them efficient scavengers of H2O2 under stress (Sharma et al. 2012). Guaiacol peroxidase (GPX, EC 1.11.1.7) is associated with many important biosynthetic processes, including cell-wall lignification, indole-3-acetic acid (IAA) degradation, ethylene biosynthesis, wound healing, and defense against stresses (Sharma et al. 2012). CAT (1.11.1.6) is present in the form of various isoforms in plant cells (Corpas et al. 2017). CAT helps in eliminating H2O2 generated during photorespiration, β-oxidation of fatty acids, and purine catabolism in peroxisomes by converting it into oxygen and water (Gill and Tuteja 2010, Corpas et al. 2017). It can directly decompose H2O2 and is indispensable for ROS detoxification during stress conditions (Gill and Tuteja 2010). In the current study, we assess the physiological features of T. ulmi galls and tissues of Ulmus pumila leaves involved in the galling process. The aim of the study was to answer the following questions: 1) Does the aphid feeding activity induce oxidative stress and alter defense mechanisms in the host plant? 2) Does the plant reaction change depending on the gall developmental stage (number of aphids feeding inside the gall)? Material and Methods Plant Material and Sampling The experimental samples were collected from U. pumila trees, which were part of urban green areas of Lublin, Poland (51.24°N, 22.57°S). Plant material was collected in three stages of gall development (treatments): 1) initial stage, 2) fully developed stage, and 3) mature gall stage. In the first stage, the galls were green, about 5 mm in height, with one fundatrix larva inside. In the second stage, the galls were green and fully developed. There was an adult fundatrix and a few young offspring inside. In the third stage, just before the opening, the galls were yellowish, mostly with nymphs in the fourth stadium and adult migrants inside. Plant material consisted of 20–50 leaves (depending on the size of the galls) with galls collected from 3–4 trees within hand’s reach. Phenologically similar intact leaves (located on the same site of the shoot without galled leaves) served as a control in each sampling point. The collected materials were kept in plastic bags and brought to the laboratory within 1 h. Subsequently, all galls were cutoff and the aphids were removed from galls by a soft brush. Parts of the galled leaf blade with visible damage were separated. The plant material was categorized as four biological replicates: 1) intact leaves, 2) undamaged part (without visible discoloration and corrugation) of the galled leaf lamina, 3) damaged part of the galled leaf lamina, and 4) galls alone. One sample (one biological replicate) contained a mix of several leaves in each category or galls to make it more representative. In each treatment (i.e., initial stage, fully developed, mature gall), four biological replicates were used in three analytical replications. Biochemical Analysis H2O2 content was estimated by forming a titanium–hydro peroxide complex (Jena and Choudhuri 1981). Plant material (50 mg fresh weight [FW]) was grinded in 3 ml of phosphorus buffer (50 mM, pH 6.5) in 4°C, and then the mixture was centrifuged at 6,000 × g for 25 min. Next, 1.5 ml of the supernatant was added to 0.5 ml TiO2 in 20% (v/v) H2SO4 and centrifuged again at 6,000 × g for 15 min at room temperature. The absorbance of the supernatant was measured at 410 nm against blank reagent with a Cecil CE 9500 spectrophotometer (Cecil Instruments, Cambridge, England). The H2O2 concentration in the sample was calculated using the molar absorbance coefficient 0.28 μM−1 cm−1 and was expressed as nanomoles per 1 g FW. Electrolyte leakage (EL) was measured to assess the injury percentage of cell membrane according to the method of Kościelniak (1993), with an Elmetron CC-317 microcomputer conductometer. Ten leaf rings of 0.9 cm diameter were cut with a cork borer from each sample. The plant material was transferred to 20 cm3 deionized water and shaken at room temperature for 24 h. Then, the initial electrical conductivity (K1) was determined and the samples were autoclaved at 100°C for 15 min; after 24 h of shaking, the final conductivity of the solution was measured again to determine the total electrolyte content (K2). EL was calculated using the formula: EL (%) = (K1/K2) 100. The level of membrane lipid peroxidation was measured as the amount of malondialdehyde determined by the content of thiobarbituric acid reactive substances (TBARS), according to the method of Heath and Packer (1968). Plant material (0.2 g) was homogenized in 0.1 M potassium phosphate buffer, pH 7.0, then centrifuged at 12,000 × g for 20 min. Two cm3 of 20% trichloroacetic acid containing 0.5% thiobarbituric acid (TBA) were added to 0.5 cm3 of the supernatant. Then the test-tubes were incubated in a water bath at 95°C for 30 min and then cooled and centrifuged again at 10,000 × g for 10 min. The absorbance was measured at 532 and 600 nm with a spectrophotometer mentioned previously. The TBARS concentration in the samples was calculated using the molar absorbance coefficient (155 nM−1 cm−1) and results are presented as nanomoles per 1 g FW. Enzyme Assays Fresh leaf tissue (0.2 g) was homogenized in a mortar and pestle with 0.05 mol dm−3 phosphate buffer (pH 7.0), containing 0.2 mol dm−3 Ethyleno-Diamine-Tetra-Acetate acid (EDTA) and 2% Poly-Vinyl-Pyrrolidone (PVP) at 4°C. The homogenate was then centrifuged at 10,000 × g for 10 min at 4°C. The thus obtained supernatant was used directly for enzyme analysis. The enzymes activity was measured using Cecil CE 9500 spectrophotometer (Cecil Instruments). Guaicol peroxidase (GPOD) activity was assayed as per method of Małolepsza et al. (1994). The assay system contained 0.5 cm3 of 0.05 mol dm−3 phosphorus buffer pH 5.6, 0.5 cm3 of 0.02 mol dm−3 guaiacol, 0.5 cm3 of 0.06 mol dm−3 H2O2, and 0.5 cm3 of enzymatic extract. Enzyme activity was measured as change of absorbance at wavelength 480 nm for 4 min at 1 min intervals. POD activity toward guaiacol was determined using the absorbance coefficient for this enzyme (26.6 mM cm−1). Its activity was expressed as a change of peroxidase activity per FW, expressed as U mg−1 FW. APX activity was assayed according to Nakano and Asada (1981). The reaction mixture contained 1.8 ml 0.1 M phosphorus buffer pH 6.0, 20 μl of 5 mM sodium ascorbate, 100 μl of 1mM H2O2, and 100 μl of enzymatic extract. Absorbance was monitored at a wavelength 290 nm for 5 min, measured at 1-min intervals. APX activity was determined using the absorbance coefficient for this enzyme (2,800 M−1 cm−1). Its activity was expressed as the change of peroxidase activity per FW, expressed as U mg−1 FW. CAT activity was determined using the protocol described by Chance and Meahly (1955) and modified by Wiloch et al. (1999). The reaction mixture contained 2 cm3 of 50 mM K-phosphorus buffer pH 7.0, 0.2 cm3 of H2O2, and 0.1 cm3 of enzymatic extract. The extinction was measured for 3 min, reading at the initial and final stage of the experiment at wavelength 240 nm. CAT activity was determined using the CAT absorbance coefficient (0.036 mM cm−1). The results were converted to CAT activity per FW, expressed as U mg−1 FW. Statistical Analysis It was reported that biochemical features of leaves changed during developmental stages from bud break to senescence (Polle et al. 2001, Jiang et al. 2006). Therefore, univariate analysis of variance was applied to analyze the effect of aphid feeding on the following variables: H2O2, EL, TBARS, APX, GPOD, CAT in galls, and different parts of elm leaves at each stage of gall development. The Tukey’s Honest Significant Difference (HSD) test was used for multiple comparisons of means. Statistical significance was set at α = 0.05. Biochemical assays were performed in three independent biological replicates (n = 3). Arithmetic means with ±SD are presented in the figures. All statistical analyses were performed using Statistica 13.1 (StatSoft, Poland). Results The Initial Period of Gall Formation (Stage I) The concentration of H2O2 in undamaged parts of galled leaves and in gall tissues was lower in this stage, as compared to intact leaves, and reached the level of 0.41 and 0.67 µmol g−1 FW, respectively. The significant increase of this molecule was found only in the damaged parts of galled leaves (Fig. 1). EL did not differ significantly in the analyzed tissues and ranged from 57.15 to 66.5% (Table 1). No lipid peroxidation, measured as TBARS, was recorded due to fundatrix larvae infestation. Even lower TBARS content was noted in gall tissues in comparison to other tissues (Fig. 3). However, fundatrix feeding clearly affected antioxidant enzyme activities. More than 2-fold increase in APX activity was observed in gall tissues and damaged parts of galled leaves in comparison to control (Fig. 4). On the other hand, GPOD activity was clearly downregulated in damaged parts of galled leaves and in gall tissues (Fig. 5). Moreover, a significant enhancement of CAT activity was detected in all tissues involved in galling process (Fig. 6). Table 1. Results of analysis of variance (ANOVA) on H2O2, EL, TBARS, APX, GPOD, and CAT measured in Tetraneura ulmi L. galls, and galled and ungalled Ulmus pumila L. leaves during three gall developmental stages Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Stage I—initial period of galling, stage II—fully developed gall, stage III—mature gall. Corresponding figure: H2O2—Fig. 1, EL—Fig. 2, TBARS—Fig. 3, APX—Fig. 4, GPOD—Fig. 5, and CAT—Fig. 6. View Large Table 1. Results of analysis of variance (ANOVA) on H2O2, EL, TBARS, APX, GPOD, and CAT measured in Tetraneura ulmi L. galls, and galled and ungalled Ulmus pumila L. leaves during three gall developmental stages Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Stage I—initial period of galling, stage II—fully developed gall, stage III—mature gall. Corresponding figure: H2O2—Fig. 1, EL—Fig. 2, TBARS—Fig. 3, APX—Fig. 4, GPOD—Fig. 5, and CAT—Fig. 6. View Large Fig. 1. View largeDownload slide H2O2 level in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 1. View largeDownload slide H2O2 level in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). The Fully Developed Galls (Stage II) The H2O2 level was similar in all analyzed tissues with the exception of undamaged parts of galled leaves, where approximately 1.5-fold increase was noted in relation to control (Fig. 1). The cell membrane injuries, based on EL, were similar in galls and nongalled leaves, but they were significantly lower in undamaged and damaged parts of galled leaves (Fig. 2). The level of lipid peroxidation was significantly elevated in damaged parts of galled leaves, and especially in galls (more than 5-fold compared to intact leaves), reaching 41.7 and 121.4 nmol g−1 FW, respectively (Fig. 3). The highest APX activity was detected in undamaged parts of galled leaves. A slight increase of its activity was measured in damaged parts of galled leaves and in gall tissues (Fig. 4). GPOD activity was similar in all analyzed tissues and did not differ from its activity in control leaves (Table 1; Fig. 5). Aphid feeding significantly suppressed CAT activity in both parts of galled leaves and in the galls (Fig. 6). Fig. 2. View largeDownload slide EL in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 2. View largeDownload slide EL in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 3. View largeDownload slide TBARS content in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 3. View largeDownload slide TBARS content in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 4. View largeDownload slide APX activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 4. View largeDownload slide APX activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 5. View largeDownload slide GPOD activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 5. View largeDownload slide GPOD activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 6. View largeDownload slide CAT in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 6. View largeDownload slide CAT in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). The Mature Galls (Stage III) The highest level of H2O2, percentage of cell membrane damage, and lipid peroxidation in gall tissues were detected at this stage. There was also a significantly higher concentration of H2O2 measured in undamaged parts of galled leaves (Fig. 1). EL in both parts of galled leaves was similar in relation to intact leaves (Fig. 2). TBARS content in galled leaves (both parts) was lower, as compared to intact leaves (Fig. 3). A similar pattern of changes was observed in the case of both peroxidases (Figs 4 and 5). Their activity was significantly lower in galled leaves, particularly in galls when compared to intact leaves. A significant increase in CAT activity was observed in both parts of galled leaves, while no activity of this enzyme was detected in galls (Fig. 6). Discussion All analyzed parameters, i.e., membrane permeability, TBARS and H2O2 content, APX, GPOD, and CAT activity were changed in gall tissues and in different parts of galled leaves during galling process. These findings indicate that T. ulmi–induced oxidative stress and altered defense mechanisms in U. pumila leaves. The plant reaction to galling aphid activity was different and depended on the gall developmental stage. It is noteworthy that, increasing number of aphids feeding inside the gall, resulted in enhanced ROS production and membrane damage associated with declining antioxidant enzyme activities in the subsequent stages of gall development. Young fundatrix larva intensively punctures bottom side of the leaf blade in the initial period of gall formation. The blade is slightly deformed between veins at the sucking site, irregularly corrugated and discolored. After several days of sucking, larva settles and feeds in one place on the abaxial face of the leaf. The gall that encloses fundatrix is rapidly formed on the opposite side of the leaf (Urban 2003, Kmieć et al. 2016). In the present study, an increased H2O2 generation was observed only in the tissues surrounding young galls. Elevated H2O2 levels together with cell death induction occur as an oxidative response to herbivore feeding as part of plant defense mechanisms (Radville et al. 2011). Martinez de Ilarduya et al. (2003) demonstrated that H2O2 production during Macrosiphum euphorbiae (Thom.) tomato infestation was limited to the feeding site, which suggested a locally restricted defense response of the plant. ROS production is one of the first steps in the hypersensitive response (HR) of plants to pathogens and, in the case of galls, may trigger gall morphogenesis (Oliveira et al. 2010, Isaias and Oliveira 2012). The low concentration of ROS may lead to biochemical alterations that modulate plant cell responses and gall development (Oliveira et al. 2016). Our study did not detect any cell membrane damage, expressed as EL and TBARS content in any tissues of galled leaves. However, elevated CAT activity in all analyzed parts of galled leaves as well as APX, but not GPOD, in young galls and damaged parts of galled leaves indicated that the antioxidant machinery in young galls and different parts of galled leaves was activated upon aphid feeding. What is more, efficient scavenging of H2O2 was present in newly developed galls. Rapid induction of APX activity in chrysanthemum infested by Macrosiphoniella sanborni (Gillette) and in winter triticale after infestation by Sitobion avenae (F.) and Rhopalosiphum padi (L.) indicated that this enzyme is involved in early responses to aphid attack (He et al. 2011, Łukasik et al. 2012). Isaias et al. (2015) proposed that cell reprogramming, occurring as a response to biotic stimuli of gall initiation and development, was affected by cell redox state, and depended on the balance between ROS production and scavenging. The growth and development of galls require the control of toxic and signaling effect of ROS in plant cell compartments. CAT and APX have been proposed as crucial for maintaining the redox balance during oxidative stress (Sharma et al. 2012, and references therein). The propagation of systemic signal in the galling process of T. ulmi might be suppressed by CAT that directly decomposes H2O2, and/or APX in combination with glutathione reductase during repeated oxidation-reduction reactions promoted by GSH (Hung et al. 2005, Suzuki and Mittler 2012). The improvement of antioxidant enzyme activities in ROS scavenging could increase the ability of plants to tolerate oxidative stress and delay its senescence. It has been established that the early stages of gall formation evoke stress responses, such as accumulation of active peroxisomes and glyoxysomes (Raman 2012). High activity of CAT and APX in this period of T. ulmi galling could be due to the fact that peroxisomes are major sites of H2O2 formation (Corpas et al. 2017). CAT scavenges H2O2 generated in this organelle during photorespiratory oxidation, β-oxidation of fatty acids and other enzyme systems, whereas APX is involved in ascorbate-glutathione cycle, which enables plants to control H2O2 content (Sharma et al. 2012, Corpas et al. 2017). Our previous study indicated an increase in polyamine contents in gall tissue during the gall initiation and at full maturity stages (Kmieć et al. 2018b). Polyamines are involved in a variety of regulatory processes, e.g., gene expression, promotion of growth, and cell division or modulation of cell signaling. They can maintain the stability of cell membrane structure and function by enhancing the activity of protective enzymes or directly by ROS scavenging (Geng et al. 2007). In the second stage of gall development, the galls were fully grown and young migrant larvae fed with adult fundatrix inside the gall. In turn, mature galls (just before “opening”) were filled mostly with migrant nymphs. Further increase in membrane permeability was observed during these periods, especially in galls. Lipid peroxidation in gall tissues was more than 5-fold higher as compared to intact leaves in the second stage and even 8-fold higher when compared to galled leaves in the last stage. Membranes of plant cells constitute a valuable storage of lipid molecules that can be mobilized and used during germination, ROS formation in senescing tissue, stress responses, pathogen, and herbivore defense (Golan et al. 2013, Mai et al. 2013, Bhattacharjee 2014, Kmieć et al. 2014). Peroxidation of membrane fatty acids in plant cells occurs in enzymatic and nonenzymatic pathways with the generation of breakdown products in the form of alcohols, ketones, alkanes, aldehydes, and ethers. Some of these products are highly reactive and can be considered as secondary toxic cell messengers. They participate in several physiological pathways, including cell death, antioxidant defense induction, and cell-signaling protein modifications (Bhattacharjee 2014). Oliveira and Isaias (2010) reported an increase in ROS production starting from young galls until senescence and related it to oxidative stress and cytological characteristics. High accumulation of ROS in mature galls of Cecidomyiidae induced on Aspidosperma spruceanum Müell.Arg. leaves was detected by Formiga and et al. (2011). The highest level of H2O2 in gall tissue at the stage of mature galls was also recorded in our study. Aphid stylets penetrate cells by symplast punctures during feeding and cause tissue injury (Tjallingii 2006). These hemipterans deceive their host plants by delivering salivary chemicals into the plant to affect wound healing, defense signaling pathways, and volatile emissions (Walling 2008). Our previous research revealed that more than 80 individuals could feed inside the mature gall (Kmieć and Kot 2007), which could explain the high level of gall oxidative stress. Aphids, as phloem parasites, can feed continuously for several days and degrade leaf proteins and utilize increased translocation of photosynthesis products (Giordanengo et al. 2010). Galls are considered to be effective physiological sinks due to their low rates of CO2 fixation. Larson and Whitham (1991) have shown that the galls of Pemphigus betae Doane act as strong sinks by reversing normal transport patterns and inducing mature leaves to import photoassimilates produced in neighboring leaves. The low content of chlorophyll a and b recorded in the T. ulmi galls suggested that the photosynthetic capacity was lower in galls than in other tissues (Kmieć et al. 2018a). Sugars drained to gall sites may play secondary roles in the dissipation of free radical species accumulated in gall tissues (Isaias et al. 2015). Elevated APX activity detected in the second stage of T. ulmi gall development, especially in undamaged parts of galled leaves, could be an attempt to delay the deleterious effects of aphid feeding. However, the generation of high levels of free radicals, lipid peroxidation, and membrane permeability in the third stage of T. ulmi galling process was associated with a decrease in both peroxidase activities and CAT inactivation in the galls. The high level of root-knot nematode and crown gall bacterial infection resulted in a significant inhibition of antioxidant enzyme activities and upregulated TBARS and H2O2 formation in grape (El-Beltagi et al. 2011). In our study, enzyme activities were also considerably lower in other tissues of galled leaves, with the exception of high CAT activity in damaged and undamaged parts of galled leaves. It is possible that enhanced CAT activity protects plant tissues from lipid peroxidation in the vicinity of the gall. Various biochemical components of plant cells and tissues exert a profound effect on insect biology. The POD activity in galled leaves and galls was lower at all experimental time points when compared to control leaves. The lack of membrane damage, low concentration of H2O2, and high antioxidant enzyme (APX and CAT) activities in tissues of galled leaves and in the galls appeared to be beneficial for the aphids during the first period of galling process, when a single fundatrix fed actively inside the galls. However, peroxidases could function as defensive proteins in the herbivore gut, because of the ability of H2O2 production (Zhu-Salzman et al. 2008). Peroxidases impair nutrient uptake by insects through the formation of electrophiles by oxidizing monohydroxyphenols or dihydroxyphenols (Gulsen et al. 2010, War et al. 2012). High activity of antioxidant enzymes in plant tissues during initial period of gall development could be a signal for fundatrix to produce winged progeny (Ogawa and Miura 2014). The increased number of young aphids in galls and their feeding capacity resulted in enhanced production of H2O2/ROS during consecutive stages of gall development, especially when the galls were mature and contained numerous migrant larvae. Aphids were subjected to exogenous H2O2 and other ROS generated by plants to defend against herbivory. ROS cause oxidative damage to the midgut cells and reduce nutrient absorption by insects (Urbańska 2009, Sytykiewicz 2011, Łukasik and Goławska 2013). However, larvae of S. avenae and R. padi cereal aphids had a higher level of nonenzymatic and enzymatic antioxidants than apterae adults (Łukasik et al. 2008). After a relatively short period of nymphal feeding, T. ulmi emerges from galls, which begin to wither, as adult winged females (Urban 2003, Kmieć et al. 2016). According to Oliveira and Isaias (2010), the cells of mature galls of Cecidomyiidae on A. spruceanum showed signs of degradation and PCD, characterized by pyknotic nuclei, loss of membrane integrity, and breakdown of the organelle membrane systems. In conclusion, we demonstrate that the process of gall initiation and development mediated by T. ulmi exposes the host plant to high oxidative stress and induces biochemical and physiological alterations. In the subsequent stages of gall development, a progressive increase in ROS production and cell membrane damage was associated with declining antioxidant enzyme activities, especially in gall tissues. The culmination of these processes took place in mature galls. The concentration of damaged cellular components and the associated loss of function induce aging and death of plant cells. The closed galls of T. ulmi, when mature, explode and release emigrants (Álvarez et al. 2013). On the other hand, gall insects exert considerable control over their host plants (Allison and Schultz 2005, Kot et al. 2017) and can be expected to minimize any host plant defensive response. In the initial phases of gall development, high levels of antioxidant enzyme activities may control the irreversible increasing of free radical levels in galls. However, T. ulmi galling process seems to be similar to the plant-HR in pathogen infection, when an increase of ROS induces cell death in damaged areas, which in consequence isolates and starves invading organisms. These findings are confirmed by changes in host leaf characteristics during galling (Kmieć et al. 2016). The stages of gall development are likely to be part of cell death that is triggered by aphid feeding. It seems that the gall is the result of a biochemical struggle between the host plant and the gall inducer. Acknowledgments Authors would like to thank two anonymous reviewers for insightful comments on the manuscript. 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Tetraneura ulmi (Hemiptera: Eriosomatinae) Induces Oxidative Stress and Alters Antioxidant Enzyme Activities in Elm Leaves

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Abstract

Abstract Gall formation is induced by an insect, which changes normal plant development and results in the formation of a new organ, following distinct stages of metabolic and developmental alterations. Research on mechanisms of recognition and responses to biotic stress may help to understand the interactions between galling aphids and their host plants. In this study, Tetraneura ulmi L. (Hemiptera: Eriosomatinae) galls and Ulmus pumila L. (Rosales: Ulmaceae) leaves were used as a model. Concentrations of hydrogen peroxide (H2O2) and thiobarbituric acid reactive substances, electrolyte leakage, as well as the activity of ascorbate peroxidase, guaiacol peroxidase, and catalase (CAT) were determined in galls and two parts of galled leaves (with and without visible damage). Biochemical analyses were performed at three stages of gall development: initial, fully developed, and mature galls. A slight increment in H2O2 content with a strong enhancement of ascorbate peroxidase and CAT activities were observed in galls and galled leaves in the first stage. In subsequent stages of gall development, a progressing increase in H2O2 production and cell membrane damage was associated with declining antioxidant enzyme activities, especially in gall tissues. The stages of gall development are likely to be part of cell death triggered by aphid feeding. It seems that the gall is the result of a biochemical struggle between the host plant and the gall inducer. aphid, biotic stress, gall development, plant defense Most insect herbivores have very intimate associations with their host plants. Galling aphids are of special interest, because they change normal plant development by stimulating tissue differentiation and new organ formation (Wool 2004). The activity of galling herbivores causes structural and biochemical alterations in host plant tissues. Initiation, increment in mass and qualitative differentiation, maturation, and senescence are key phases of gall development (Raman 2011). The isolation and insulation of one or a few cells on the plant from their normal course of differentiation is the earliest event in gall induction. These cells are irreversibly changed by an external stimulus from the inducing insect (Raman 2012). Aphid galls are formed only on young, growing plant tissues, and their colonization activity is timed with the host plant phenology (Wool 2004, Oliveira et al. 2016). The Tetraneura ulmi L. aphid forms bean-shaped galls on young leaves of different elm species (Ulmus sp.) in the spring (Kmieć and Kot 2007, Blackman and Eastop 2018). Each gall is induced by a single female (fundatrix) emerging from fertilized egg. Aphid propagates in the gall by parthenogenetic reproduction. Wingless progeny becomes winged in June and disperses from the cracking gall to find the secondary host. Only two generations of aphids develop in the gall (Urban 2003, Kmieć and Kot 2007). Aphids use their slender and flexible stylets to penetrate plant tissue intercellularly to access the sieve elements for feeding (Walling 2008, Giordanengo et al. 2010). However, occasionally, stylets also puncture cells and minute amounts of cell contents are ingested (Tjallingii 2006). During feeding, insects introduce effectors that alter plant defense signaling, infestation symptoms, and plant development (Giordanengo et al. 2010, Rodriguez and Bos 2013). However, no cecidogenic compounds were identified in aphid saliva; hypertrophy-inducing triacylglycerides were isolated from Colopha moriokaensis (Monz.) body (Ohta et al. 2000). Although aphids feed primarily from the phloem, the identified effectors could also mediate plant–aphid interactions in other cell types or in intercellular spaces (Will et al. 2013). Plants have the ability to respond to insect infestation by changing their biochemical states. Early defense responses to insect feeding include protein phosphorylation, membrane depolarization, calcium influx, and release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2; Maffei et al. 2007, Giordanengo et al. 2010). A low level of ROS is maintained by the presence of various secondary plant metabolites and scavenging enzymes under normal plant developmental conditions (Gill and Tuteja 2010). According to Giordanengo et al. (2010) and Mai et al. (2013), aphid feeding induces oxidative burst and an increase in ROS production in the host plant. Despite their potential toxicity, ROS in fact play a dual role in vivo (Sharma et al. 2012). Lower doses of ROS are employed as signals that mediate at least part of the stress responses (Hung et al. 2005), while at higher concentrations, they are part of the direct defense and pose a significant threat that may eventually lead to programed cell death (PCD) (Sharma et al. 2012). ROS can provoke reversible or irreversible modifications of proteins, causing alterations in the regulation of plant metabolism and activation of transcriptional processes (Gill and Tuteja 2010). H2O2 is a strongly depolarizing molecule among ROS that can be induced by the feeding insect. H2O2 levels increase in response to herbivores as long as the attacks persist (Maffei 2007). An increase in ROS production is accompanied by higher lipid peroxidation under stress conditions. Degradation of polyunsaturated fatty acids can lead to chain breakage, thereby increasing membrane fluidity and permeability (Sharma et al. 2012, Bhattacharjee 2014). Efficient enzymatic and nonenzymatic defense plant system are activated to cope with the elevated ROS levels (Maffei et al. 2007). Peroxidases and catalase (CAT) are among the enzymes that play an important role in plant stress caused by insect feeding. The presence of oxidative enzymes at H2O2 generation sites can serve to detoxify ROS that accumulate in response to abiotic and biotic stresses (War et al. 2012). Ascorbate peroxidase (APX, EC 1.1.11.1) is regarded as one of the most widely distributed antioxidant enzymes in plant cells, and APX isoforms have significantly higher affinity for H2O2 than CAT, making them efficient scavengers of H2O2 under stress (Sharma et al. 2012). Guaiacol peroxidase (GPX, EC 1.11.1.7) is associated with many important biosynthetic processes, including cell-wall lignification, indole-3-acetic acid (IAA) degradation, ethylene biosynthesis, wound healing, and defense against stresses (Sharma et al. 2012). CAT (1.11.1.6) is present in the form of various isoforms in plant cells (Corpas et al. 2017). CAT helps in eliminating H2O2 generated during photorespiration, β-oxidation of fatty acids, and purine catabolism in peroxisomes by converting it into oxygen and water (Gill and Tuteja 2010, Corpas et al. 2017). It can directly decompose H2O2 and is indispensable for ROS detoxification during stress conditions (Gill and Tuteja 2010). In the current study, we assess the physiological features of T. ulmi galls and tissues of Ulmus pumila leaves involved in the galling process. The aim of the study was to answer the following questions: 1) Does the aphid feeding activity induce oxidative stress and alter defense mechanisms in the host plant? 2) Does the plant reaction change depending on the gall developmental stage (number of aphids feeding inside the gall)? Material and Methods Plant Material and Sampling The experimental samples were collected from U. pumila trees, which were part of urban green areas of Lublin, Poland (51.24°N, 22.57°S). Plant material was collected in three stages of gall development (treatments): 1) initial stage, 2) fully developed stage, and 3) mature gall stage. In the first stage, the galls were green, about 5 mm in height, with one fundatrix larva inside. In the second stage, the galls were green and fully developed. There was an adult fundatrix and a few young offspring inside. In the third stage, just before the opening, the galls were yellowish, mostly with nymphs in the fourth stadium and adult migrants inside. Plant material consisted of 20–50 leaves (depending on the size of the galls) with galls collected from 3–4 trees within hand’s reach. Phenologically similar intact leaves (located on the same site of the shoot without galled leaves) served as a control in each sampling point. The collected materials were kept in plastic bags and brought to the laboratory within 1 h. Subsequently, all galls were cutoff and the aphids were removed from galls by a soft brush. Parts of the galled leaf blade with visible damage were separated. The plant material was categorized as four biological replicates: 1) intact leaves, 2) undamaged part (without visible discoloration and corrugation) of the galled leaf lamina, 3) damaged part of the galled leaf lamina, and 4) galls alone. One sample (one biological replicate) contained a mix of several leaves in each category or galls to make it more representative. In each treatment (i.e., initial stage, fully developed, mature gall), four biological replicates were used in three analytical replications. Biochemical Analysis H2O2 content was estimated by forming a titanium–hydro peroxide complex (Jena and Choudhuri 1981). Plant material (50 mg fresh weight [FW]) was grinded in 3 ml of phosphorus buffer (50 mM, pH 6.5) in 4°C, and then the mixture was centrifuged at 6,000 × g for 25 min. Next, 1.5 ml of the supernatant was added to 0.5 ml TiO2 in 20% (v/v) H2SO4 and centrifuged again at 6,000 × g for 15 min at room temperature. The absorbance of the supernatant was measured at 410 nm against blank reagent with a Cecil CE 9500 spectrophotometer (Cecil Instruments, Cambridge, England). The H2O2 concentration in the sample was calculated using the molar absorbance coefficient 0.28 μM−1 cm−1 and was expressed as nanomoles per 1 g FW. Electrolyte leakage (EL) was measured to assess the injury percentage of cell membrane according to the method of Kościelniak (1993), with an Elmetron CC-317 microcomputer conductometer. Ten leaf rings of 0.9 cm diameter were cut with a cork borer from each sample. The plant material was transferred to 20 cm3 deionized water and shaken at room temperature for 24 h. Then, the initial electrical conductivity (K1) was determined and the samples were autoclaved at 100°C for 15 min; after 24 h of shaking, the final conductivity of the solution was measured again to determine the total electrolyte content (K2). EL was calculated using the formula: EL (%) = (K1/K2) 100. The level of membrane lipid peroxidation was measured as the amount of malondialdehyde determined by the content of thiobarbituric acid reactive substances (TBARS), according to the method of Heath and Packer (1968). Plant material (0.2 g) was homogenized in 0.1 M potassium phosphate buffer, pH 7.0, then centrifuged at 12,000 × g for 20 min. Two cm3 of 20% trichloroacetic acid containing 0.5% thiobarbituric acid (TBA) were added to 0.5 cm3 of the supernatant. Then the test-tubes were incubated in a water bath at 95°C for 30 min and then cooled and centrifuged again at 10,000 × g for 10 min. The absorbance was measured at 532 and 600 nm with a spectrophotometer mentioned previously. The TBARS concentration in the samples was calculated using the molar absorbance coefficient (155 nM−1 cm−1) and results are presented as nanomoles per 1 g FW. Enzyme Assays Fresh leaf tissue (0.2 g) was homogenized in a mortar and pestle with 0.05 mol dm−3 phosphate buffer (pH 7.0), containing 0.2 mol dm−3 Ethyleno-Diamine-Tetra-Acetate acid (EDTA) and 2% Poly-Vinyl-Pyrrolidone (PVP) at 4°C. The homogenate was then centrifuged at 10,000 × g for 10 min at 4°C. The thus obtained supernatant was used directly for enzyme analysis. The enzymes activity was measured using Cecil CE 9500 spectrophotometer (Cecil Instruments). Guaicol peroxidase (GPOD) activity was assayed as per method of Małolepsza et al. (1994). The assay system contained 0.5 cm3 of 0.05 mol dm−3 phosphorus buffer pH 5.6, 0.5 cm3 of 0.02 mol dm−3 guaiacol, 0.5 cm3 of 0.06 mol dm−3 H2O2, and 0.5 cm3 of enzymatic extract. Enzyme activity was measured as change of absorbance at wavelength 480 nm for 4 min at 1 min intervals. POD activity toward guaiacol was determined using the absorbance coefficient for this enzyme (26.6 mM cm−1). Its activity was expressed as a change of peroxidase activity per FW, expressed as U mg−1 FW. APX activity was assayed according to Nakano and Asada (1981). The reaction mixture contained 1.8 ml 0.1 M phosphorus buffer pH 6.0, 20 μl of 5 mM sodium ascorbate, 100 μl of 1mM H2O2, and 100 μl of enzymatic extract. Absorbance was monitored at a wavelength 290 nm for 5 min, measured at 1-min intervals. APX activity was determined using the absorbance coefficient for this enzyme (2,800 M−1 cm−1). Its activity was expressed as the change of peroxidase activity per FW, expressed as U mg−1 FW. CAT activity was determined using the protocol described by Chance and Meahly (1955) and modified by Wiloch et al. (1999). The reaction mixture contained 2 cm3 of 50 mM K-phosphorus buffer pH 7.0, 0.2 cm3 of H2O2, and 0.1 cm3 of enzymatic extract. The extinction was measured for 3 min, reading at the initial and final stage of the experiment at wavelength 240 nm. CAT activity was determined using the CAT absorbance coefficient (0.036 mM cm−1). The results were converted to CAT activity per FW, expressed as U mg−1 FW. Statistical Analysis It was reported that biochemical features of leaves changed during developmental stages from bud break to senescence (Polle et al. 2001, Jiang et al. 2006). Therefore, univariate analysis of variance was applied to analyze the effect of aphid feeding on the following variables: H2O2, EL, TBARS, APX, GPOD, CAT in galls, and different parts of elm leaves at each stage of gall development. The Tukey’s Honest Significant Difference (HSD) test was used for multiple comparisons of means. Statistical significance was set at α = 0.05. Biochemical assays were performed in three independent biological replicates (n = 3). Arithmetic means with ±SD are presented in the figures. All statistical analyses were performed using Statistica 13.1 (StatSoft, Poland). Results The Initial Period of Gall Formation (Stage I) The concentration of H2O2 in undamaged parts of galled leaves and in gall tissues was lower in this stage, as compared to intact leaves, and reached the level of 0.41 and 0.67 µmol g−1 FW, respectively. The significant increase of this molecule was found only in the damaged parts of galled leaves (Fig. 1). EL did not differ significantly in the analyzed tissues and ranged from 57.15 to 66.5% (Table 1). No lipid peroxidation, measured as TBARS, was recorded due to fundatrix larvae infestation. Even lower TBARS content was noted in gall tissues in comparison to other tissues (Fig. 3). However, fundatrix feeding clearly affected antioxidant enzyme activities. More than 2-fold increase in APX activity was observed in gall tissues and damaged parts of galled leaves in comparison to control (Fig. 4). On the other hand, GPOD activity was clearly downregulated in damaged parts of galled leaves and in gall tissues (Fig. 5). Moreover, a significant enhancement of CAT activity was detected in all tissues involved in galling process (Fig. 6). Table 1. Results of analysis of variance (ANOVA) on H2O2, EL, TBARS, APX, GPOD, and CAT measured in Tetraneura ulmi L. galls, and galled and ungalled Ulmus pumila L. leaves during three gall developmental stages Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Stage I—initial period of galling, stage II—fully developed gall, stage III—mature gall. Corresponding figure: H2O2—Fig. 1, EL—Fig. 2, TBARS—Fig. 3, APX—Fig. 4, GPOD—Fig. 5, and CAT—Fig. 6. View Large Table 1. Results of analysis of variance (ANOVA) on H2O2, EL, TBARS, APX, GPOD, and CAT measured in Tetraneura ulmi L. galls, and galled and ungalled Ulmus pumila L. leaves during three gall developmental stages Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Source  df  Stage I  Stage II  Stage III  F  P  F  P  F  P  H2O2  3,8  42.791  ˂0.001  607.130  ˂0.001  126.204  ˂0.001  EL  3,8  1.249  0.35  24.280  ˂0.001  59.266  ˂0.001  TBARS  3,8  20.55639  ˂0.001  3081.870  ˂0.001  699.6260  ˂0.001  APX  3,8  23.150  ˂0.001  420.050  ˂0.001  147.651  ˂0.001  GPOD  3,8  22.700  ˂0.001  3.520  0.068  102.876  ˂0.001  CAT  3,8  36.612  ˂0.001  17.120  0.001  788.102  ˂0.001  Stage I—initial period of galling, stage II—fully developed gall, stage III—mature gall. Corresponding figure: H2O2—Fig. 1, EL—Fig. 2, TBARS—Fig. 3, APX—Fig. 4, GPOD—Fig. 5, and CAT—Fig. 6. View Large Fig. 1. View largeDownload slide H2O2 level in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 1. View largeDownload slide H2O2 level in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). The Fully Developed Galls (Stage II) The H2O2 level was similar in all analyzed tissues with the exception of undamaged parts of galled leaves, where approximately 1.5-fold increase was noted in relation to control (Fig. 1). The cell membrane injuries, based on EL, were similar in galls and nongalled leaves, but they were significantly lower in undamaged and damaged parts of galled leaves (Fig. 2). The level of lipid peroxidation was significantly elevated in damaged parts of galled leaves, and especially in galls (more than 5-fold compared to intact leaves), reaching 41.7 and 121.4 nmol g−1 FW, respectively (Fig. 3). The highest APX activity was detected in undamaged parts of galled leaves. A slight increase of its activity was measured in damaged parts of galled leaves and in gall tissues (Fig. 4). GPOD activity was similar in all analyzed tissues and did not differ from its activity in control leaves (Table 1; Fig. 5). Aphid feeding significantly suppressed CAT activity in both parts of galled leaves and in the galls (Fig. 6). Fig. 2. View largeDownload slide EL in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 2. View largeDownload slide EL in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 3. View largeDownload slide TBARS content in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 3. View largeDownload slide TBARS content in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 4. View largeDownload slide APX activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 4. View largeDownload slide APX activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 5. View largeDownload slide GPOD activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 5. View largeDownload slide GPOD activity in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 6. View largeDownload slide CAT in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). Fig. 6. View largeDownload slide CAT in Ulmus pumila L. tissues during developmental stages of Tetraneura ulmi L. galls (means ± SD). Stage number indicate: I—initial period of galling, II—fully developed gall, III—mature gall; leaves UP—undamaged part of galled leaf lamina, leaves DP—damaged part of galled leaf lamina (with visible discoloration and corrugation). Bars sharing the same letter at each stage do not differ significantly at P ≥ 0.05 (Tukey’s HSD test). The Mature Galls (Stage III) The highest level of H2O2, percentage of cell membrane damage, and lipid peroxidation in gall tissues were detected at this stage. There was also a significantly higher concentration of H2O2 measured in undamaged parts of galled leaves (Fig. 1). EL in both parts of galled leaves was similar in relation to intact leaves (Fig. 2). TBARS content in galled leaves (both parts) was lower, as compared to intact leaves (Fig. 3). A similar pattern of changes was observed in the case of both peroxidases (Figs 4 and 5). Their activity was significantly lower in galled leaves, particularly in galls when compared to intact leaves. A significant increase in CAT activity was observed in both parts of galled leaves, while no activity of this enzyme was detected in galls (Fig. 6). Discussion All analyzed parameters, i.e., membrane permeability, TBARS and H2O2 content, APX, GPOD, and CAT activity were changed in gall tissues and in different parts of galled leaves during galling process. These findings indicate that T. ulmi–induced oxidative stress and altered defense mechanisms in U. pumila leaves. The plant reaction to galling aphid activity was different and depended on the gall developmental stage. It is noteworthy that, increasing number of aphids feeding inside the gall, resulted in enhanced ROS production and membrane damage associated with declining antioxidant enzyme activities in the subsequent stages of gall development. Young fundatrix larva intensively punctures bottom side of the leaf blade in the initial period of gall formation. The blade is slightly deformed between veins at the sucking site, irregularly corrugated and discolored. After several days of sucking, larva settles and feeds in one place on the abaxial face of the leaf. The gall that encloses fundatrix is rapidly formed on the opposite side of the leaf (Urban 2003, Kmieć et al. 2016). In the present study, an increased H2O2 generation was observed only in the tissues surrounding young galls. Elevated H2O2 levels together with cell death induction occur as an oxidative response to herbivore feeding as part of plant defense mechanisms (Radville et al. 2011). Martinez de Ilarduya et al. (2003) demonstrated that H2O2 production during Macrosiphum euphorbiae (Thom.) tomato infestation was limited to the feeding site, which suggested a locally restricted defense response of the plant. ROS production is one of the first steps in the hypersensitive response (HR) of plants to pathogens and, in the case of galls, may trigger gall morphogenesis (Oliveira et al. 2010, Isaias and Oliveira 2012). The low concentration of ROS may lead to biochemical alterations that modulate plant cell responses and gall development (Oliveira et al. 2016). Our study did not detect any cell membrane damage, expressed as EL and TBARS content in any tissues of galled leaves. However, elevated CAT activity in all analyzed parts of galled leaves as well as APX, but not GPOD, in young galls and damaged parts of galled leaves indicated that the antioxidant machinery in young galls and different parts of galled leaves was activated upon aphid feeding. What is more, efficient scavenging of H2O2 was present in newly developed galls. Rapid induction of APX activity in chrysanthemum infested by Macrosiphoniella sanborni (Gillette) and in winter triticale after infestation by Sitobion avenae (F.) and Rhopalosiphum padi (L.) indicated that this enzyme is involved in early responses to aphid attack (He et al. 2011, Łukasik et al. 2012). Isaias et al. (2015) proposed that cell reprogramming, occurring as a response to biotic stimuli of gall initiation and development, was affected by cell redox state, and depended on the balance between ROS production and scavenging. The growth and development of galls require the control of toxic and signaling effect of ROS in plant cell compartments. CAT and APX have been proposed as crucial for maintaining the redox balance during oxidative stress (Sharma et al. 2012, and references therein). The propagation of systemic signal in the galling process of T. ulmi might be suppressed by CAT that directly decomposes H2O2, and/or APX in combination with glutathione reductase during repeated oxidation-reduction reactions promoted by GSH (Hung et al. 2005, Suzuki and Mittler 2012). The improvement of antioxidant enzyme activities in ROS scavenging could increase the ability of plants to tolerate oxidative stress and delay its senescence. It has been established that the early stages of gall formation evoke stress responses, such as accumulation of active peroxisomes and glyoxysomes (Raman 2012). High activity of CAT and APX in this period of T. ulmi galling could be due to the fact that peroxisomes are major sites of H2O2 formation (Corpas et al. 2017). CAT scavenges H2O2 generated in this organelle during photorespiratory oxidation, β-oxidation of fatty acids and other enzyme systems, whereas APX is involved in ascorbate-glutathione cycle, which enables plants to control H2O2 content (Sharma et al. 2012, Corpas et al. 2017). Our previous study indicated an increase in polyamine contents in gall tissue during the gall initiation and at full maturity stages (Kmieć et al. 2018b). Polyamines are involved in a variety of regulatory processes, e.g., gene expression, promotion of growth, and cell division or modulation of cell signaling. They can maintain the stability of cell membrane structure and function by enhancing the activity of protective enzymes or directly by ROS scavenging (Geng et al. 2007). In the second stage of gall development, the galls were fully grown and young migrant larvae fed with adult fundatrix inside the gall. In turn, mature galls (just before “opening”) were filled mostly with migrant nymphs. Further increase in membrane permeability was observed during these periods, especially in galls. Lipid peroxidation in gall tissues was more than 5-fold higher as compared to intact leaves in the second stage and even 8-fold higher when compared to galled leaves in the last stage. Membranes of plant cells constitute a valuable storage of lipid molecules that can be mobilized and used during germination, ROS formation in senescing tissue, stress responses, pathogen, and herbivore defense (Golan et al. 2013, Mai et al. 2013, Bhattacharjee 2014, Kmieć et al. 2014). Peroxidation of membrane fatty acids in plant cells occurs in enzymatic and nonenzymatic pathways with the generation of breakdown products in the form of alcohols, ketones, alkanes, aldehydes, and ethers. Some of these products are highly reactive and can be considered as secondary toxic cell messengers. They participate in several physiological pathways, including cell death, antioxidant defense induction, and cell-signaling protein modifications (Bhattacharjee 2014). Oliveira and Isaias (2010) reported an increase in ROS production starting from young galls until senescence and related it to oxidative stress and cytological characteristics. High accumulation of ROS in mature galls of Cecidomyiidae induced on Aspidosperma spruceanum Müell.Arg. leaves was detected by Formiga and et al. (2011). The highest level of H2O2 in gall tissue at the stage of mature galls was also recorded in our study. Aphid stylets penetrate cells by symplast punctures during feeding and cause tissue injury (Tjallingii 2006). These hemipterans deceive their host plants by delivering salivary chemicals into the plant to affect wound healing, defense signaling pathways, and volatile emissions (Walling 2008). Our previous research revealed that more than 80 individuals could feed inside the mature gall (Kmieć and Kot 2007), which could explain the high level of gall oxidative stress. Aphids, as phloem parasites, can feed continuously for several days and degrade leaf proteins and utilize increased translocation of photosynthesis products (Giordanengo et al. 2010). Galls are considered to be effective physiological sinks due to their low rates of CO2 fixation. Larson and Whitham (1991) have shown that the galls of Pemphigus betae Doane act as strong sinks by reversing normal transport patterns and inducing mature leaves to import photoassimilates produced in neighboring leaves. The low content of chlorophyll a and b recorded in the T. ulmi galls suggested that the photosynthetic capacity was lower in galls than in other tissues (Kmieć et al. 2018a). Sugars drained to gall sites may play secondary roles in the dissipation of free radical species accumulated in gall tissues (Isaias et al. 2015). Elevated APX activity detected in the second stage of T. ulmi gall development, especially in undamaged parts of galled leaves, could be an attempt to delay the deleterious effects of aphid feeding. However, the generation of high levels of free radicals, lipid peroxidation, and membrane permeability in the third stage of T. ulmi galling process was associated with a decrease in both peroxidase activities and CAT inactivation in the galls. The high level of root-knot nematode and crown gall bacterial infection resulted in a significant inhibition of antioxidant enzyme activities and upregulated TBARS and H2O2 formation in grape (El-Beltagi et al. 2011). In our study, enzyme activities were also considerably lower in other tissues of galled leaves, with the exception of high CAT activity in damaged and undamaged parts of galled leaves. It is possible that enhanced CAT activity protects plant tissues from lipid peroxidation in the vicinity of the gall. Various biochemical components of plant cells and tissues exert a profound effect on insect biology. The POD activity in galled leaves and galls was lower at all experimental time points when compared to control leaves. The lack of membrane damage, low concentration of H2O2, and high antioxidant enzyme (APX and CAT) activities in tissues of galled leaves and in the galls appeared to be beneficial for the aphids during the first period of galling process, when a single fundatrix fed actively inside the galls. However, peroxidases could function as defensive proteins in the herbivore gut, because of the ability of H2O2 production (Zhu-Salzman et al. 2008). Peroxidases impair nutrient uptake by insects through the formation of electrophiles by oxidizing monohydroxyphenols or dihydroxyphenols (Gulsen et al. 2010, War et al. 2012). High activity of antioxidant enzymes in plant tissues during initial period of gall development could be a signal for fundatrix to produce winged progeny (Ogawa and Miura 2014). The increased number of young aphids in galls and their feeding capacity resulted in enhanced production of H2O2/ROS during consecutive stages of gall development, especially when the galls were mature and contained numerous migrant larvae. Aphids were subjected to exogenous H2O2 and other ROS generated by plants to defend against herbivory. ROS cause oxidative damage to the midgut cells and reduce nutrient absorption by insects (Urbańska 2009, Sytykiewicz 2011, Łukasik and Goławska 2013). However, larvae of S. avenae and R. padi cereal aphids had a higher level of nonenzymatic and enzymatic antioxidants than apterae adults (Łukasik et al. 2008). After a relatively short period of nymphal feeding, T. ulmi emerges from galls, which begin to wither, as adult winged females (Urban 2003, Kmieć et al. 2016). According to Oliveira and Isaias (2010), the cells of mature galls of Cecidomyiidae on A. spruceanum showed signs of degradation and PCD, characterized by pyknotic nuclei, loss of membrane integrity, and breakdown of the organelle membrane systems. In conclusion, we demonstrate that the process of gall initiation and development mediated by T. ulmi exposes the host plant to high oxidative stress and induces biochemical and physiological alterations. In the subsequent stages of gall development, a progressive increase in ROS production and cell membrane damage was associated with declining antioxidant enzyme activities, especially in gall tissues. The culmination of these processes took place in mature galls. The concentration of damaged cellular components and the associated loss of function induce aging and death of plant cells. The closed galls of T. ulmi, when mature, explode and release emigrants (Álvarez et al. 2013). On the other hand, gall insects exert considerable control over their host plants (Allison and Schultz 2005, Kot et al. 2017) and can be expected to minimize any host plant defensive response. In the initial phases of gall development, high levels of antioxidant enzyme activities may control the irreversible increasing of free radical levels in galls. However, T. ulmi galling process seems to be similar to the plant-HR in pathogen infection, when an increase of ROS induces cell death in damaged areas, which in consequence isolates and starves invading organisms. These findings are confirmed by changes in host leaf characteristics during galling (Kmieć et al. 2016). The stages of gall development are likely to be part of cell death that is triggered by aphid feeding. It seems that the gall is the result of a biochemical struggle between the host plant and the gall inducer. Acknowledgments Authors would like to thank two anonymous reviewers for insightful comments on the manuscript. 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