Effects of aphids Myzus persicae on the changes of Ca²⁺ and H₂O₂ flux and enzyme activities in tobacco

Abstract

In order to understand the continuous defense reactions of host plants against insect attack, a tobacco variety G140 was infested by tobacco aphid Myzus persicae for 2 h to 5 d. The changes of transmembrane ionic fluxes (Ca²⁺) and hydrogen peroxide (H₂O₂) were detected by the technique of noninvasive micro-test and their relationship was further studied. It was found that H₂O₂ accumulation depended on Ca²⁺ influx. Ca²⁺ flux exhibited a strong influx at all infestation periods by aphids, while H₂O₂ showed an efflux behavior. The slight variation tendency of Ca²⁺ influx and H₂O₂ efflux was consistent. The activities of the corresponding defense proteins, peroxidase (POD) and catalase (CAT) enzyme, were enhanced to respond to the insect attacks, much higher than those tobacco in control. The Ca²⁺ influx and H₂O₂ efflux, as well as the activities of POD and CAT enzymes, were increased in a long period of aphid feeding. It indicated that a continuous physiological response of tobacco to aphid infestation could be initiated and lasted for a long time.

Keywords:

• Myzus persicae
• noninvasive micro-test technique
• peroxidase
• catalase

Introduction

Intricate defense systems were amazingly developed by plants in their long evolution process to defend attacks from insects and pathogens. It has been well known that host plants can initiate signal transduction responding to insect feeding, and then activate the related physiological and biochemical reactions. The early signal events during defense responses in plants usually include transmembrane ionic fluxes and the production of reactive oxygen species (ROS). These two events have been received considerable attentions from various research fields (Maffei et al. 2006; Howe & Jander 2008; Wu & Baldwin 2009, 2010; Roy & Dhandapani 2011). For the ionic fluxes, calcium ion (Ca²⁺) has been considered as an important second messenger in plant cells because it can be involved in many signal transduction pathways. Ca²⁺ transmembrane transport, as well as hydrogen peroxide (H₂O₂), plays an important role in triggering and regulating plant defense responses for insect attacks. Previous studies unraveled that Ca²⁺ and H₂O₂ concentrations would be significantly changed in host plants as negative effects of insects. For example, Spodoptera littoralis is able to induce Ca²⁺ influx changes at the feeding site of lima beans Phaseolus lunatus (Maffei et al. 2004). Except physical injuries, Spodoptera littoralis feeding would cause H₂O₂ accumulation near the feeding site in the injured leaves of host plants. Moreover, the accumulated H₂O₂ would diffuse into other tissues through veins and membrane in cells. The Ca²⁺ concentration was found to be increased with an increase of H₂O₂ concentration (Maffei et al. 2006). It was the larval oral secretion from orgyia ericae that induced Ca²⁺ influx and H₂O₂ accumulation in Ammopiptanthus mongolicus cells. The emergence of Ca²⁺ influx and the variation of hydrogen peroxide were considered as early signaling events in response to the regurgitated components (Gao et al. 2012).

Defense proteins, such as phenylalanine ammonia lyase, peroxidase (POD), catalase (CAT), and polyphenol oxidase, play an important role in defense reactions of host plants. They can stimulate the transport of insect-resistance signals during the defense response and induce to produce related compounds and enzymes in following cascade reactions after insect invasion (Mao et al. 2011, 2012). It has been widely reported that insect feeding influences the activities of plant defense enzymes and associates with host resistance (Zhang et al. 2012).

The noninvasive micro-test technique (NMT) is a useful tool to study membrane-transport processes of ions and molecules in plants. The main advantages of the tool were illustrated in literatures (Yin et al. 2006), including no special treatment of samples, real-time measurement of concentration and flux velocity of molecules and ions, and high resolution. In the recent two decades, this technique has been widely used to elucidate the critical roles of membrane-transport processes in response to various abiotic and biotic stressors including salinity, osmotic stress, extreme temperature, acidity, oxygen deprivation, nutritional disorder, oxidative stress, and pathogens (Shabala & Bose 2012).

Tobacco aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) is a kind of agricultural pest threatening many crops, such as tobacco (Nicotiana tabacum), potato (Solanum tuberosum), and peach tree (Amygdalus persica), around the world (Blackman & Eastop 2000). Tobacco aphid not only endangers plant hosts by directly feeding on them, but also acts as a vector for transmitting many viruses among plants (Ng & Perry 2004). Kusnierczyk et al. (2007, 2008) systemically studied the time-dependent aspect of induced changes in the aphid feeding on plants and addressed a model of plant–aphid interactions at the early phase of infestation.

Many studies on the relationship between insects and host plants focus on physical, chemical, and molecular mechanisms (Will & van Bel 2006; Roy & Dhandapani 2011; Smith & Clement 2012). The ion and molecule changes in defense response of host plants were mainly measured at discrete times. However, the continuous changes could not be determined during the feeding periods of aphids. In this work, the duration of the continuous defense reactions of host plants against aphid attack was investigated through the detection of the Ca²⁺ and H₂O₂ changes by the NMT method. The related enzymes activities in tobacco at different aphids feeding periods further unraveled the defense responses of the infested tobacco.

Materials and methods

Plants and insects

A tobacco variety G140 Nicotiana tabacum, which is prone to aphid feeding, was used. Tobacco seedlings were planted in pots (60 mm in diameter) and treated at seven or eight leaf stages. Tobacco aphids were collected from tobacco fields in Xichang city, Sichuan province, China and maintained on G140 tobacco in artificial climatic chambers at conditions of 25 ± 1°C, 75 ± 5% RH, and a photoperiod of 14:10 h (L:D).

Sample treatment

The materials for NMT and enzymatic activity measurements were treated as the following procedure. Ten wingless adult aphids were incubated to the second or the third unfurling leaf of the tobacco seedlings, which were selected with uniform sizes. The aphid-infested tobacco plants were covered by nylon mesh cages to prevent aphid escape. Those aphids were found to stay on the chosen leaf during the whole observation period. The uninfested control (CK) plants were also covered by nylon mesh cages. The infestation treatment was lasted for 0 h (CK), 2 h, 15 h, and 5 d for NMT measurement and 0 d (CK), 1 d, 2 d, and 3 d for enzymatic activity measurements. Six and three parallel samples under each treatment condition were used for NMT and enzymatic activity measurements, respectively.

The following further treatment is for NMT measurement samples. The main vein was abscised from leaves after aphid feeding. The selected leaf sample was about 4 cm². The lower epidermis at the feeding site was removed and the mesophyll cells were exposed. Prior to measurement, the mesophyll segments were placed in a Petri dish containing 10 mL of measuring solution, which was composed of 0.1 mM CaCl₂, 0.1 mM KCl, and 0.3 mM MES with pH of 6.0, for at least 20 min to minimize wounding effects. The leaf samples were then placed in another Petri dish containing the fresh measuring solution before measurement.

The following further treatment is for enzymatic activity measurement samples. The main vein of the fourth unfurling leaf, which was not infested by aphids, was abscised to avoid the contamination of POD and CAT secreted by aphid mouthparts. A leaf sample without main vein of 0.3 g was quick frozen by liquid nitrogen and stored in a refrigerator at –70°C to be determined.

Net Ca²⁺ flux measurements

Net Ca²⁺ fluxes were measured in the YoungerUSA (Xuyue Beijing) NMT Service Center by using the NMT-100 (YoungerUSA, LLC, Amherst, MA 01002, USA) with ASET 2.0 (Science Wares, Falmouth, MA 02540, USA) and iFluxes 1.0 (Younger, USA) software.

Ca²⁺-selective microelectrodes were cleaned as following procedure before each NMT test to ensure the resolution. Silanized glass microelectrodes (Φ5 ± 1 μm, XY-DJ-01, YoungerUSA) were first backfilled with 100 mM CaCl₂ to approximately 1 cm in length and then front-filled with approximately 25 μm Ca²⁺ liquid ion exchange (Ca²⁺ LIX, XY-SJ-Ca, YoungerUSA). An Ag/AgCl wire electrode holder (XY-DJGD, YoungerUSA) was inserted into the back of electrode to contact with electrolyte solution. YG003-Y05 (YoungerUSA) was used as the reference electrode (Sun et al. 2010).

Prior to flux measurements, Ca²⁺ microelectrodes were calibrated by culture media with three Ca²⁺ concentrations of 0.05 mM, 0.1 mM, and 0.5 mM. The electrodes with a Nernstian slope >26 mV/decade were used in experiments. The same calibration procedure was carried out again for the microelectrode after each test. The microelectrode was vertically positioned about 400 μm above the cell for 5 min to record the background signal (blank), and then the microelectrode was moved and positioned about 2 μm from the cell membrane to record Ca²⁺ fluxes. Each sample was measured for 10 minutes.

Net hydrogen peroxide (H₂O₂) flux measurements

The hydrogen peroxide flux in mesophyll cells was measured by the same NMT system. The Pt/Ir polarographic hydrogen peroxide microelectrodes (tip diameter 2–4 μm, XY-DJ-502, YoungerUSA) were used for detecting dissolved hydrogen peroxide in +600 mV polarization voltage as described earlier (Twig et al. 2001). YG003-Y05 (YoungerUSA) was used as the reference electrode. The hydrogen peroxide solutions with concentrations of 0.1 mM, 1 mM, and 10 mM were used to calibrate microelectrodes and the calibration procedure was the same as that in Ca²⁺ flux measurement.

Flux data analysis

Net Ca²⁺ and hydrogen peroxide fluxes were calculated by Fick’s first law of diffusion:

where J is the Ca²⁺ or hydrogen peroxide flux, D₀ is Ca²⁺ or hydrogen peroxide diffusion coefficient, dx is the distance between two points of the microelectrode repeated vibration perpendicular to surfaces of the samples, and dc is the difference of Ca²⁺ flux or hydrogen peroxide concentration between the two points. JCal V3.2 was used to calculate the net Ca²⁺ and hydrogen peroxide fluxes.

Measurements of POD and CAT activities

POD and CAT activities were measured according to the previous method in literatures (Mao et al. 2011, 2012). The unit of POD and CAT activity was defined as a 0.01 increment of OD₄₇₀ and OD₂₄₀ of fresh weight per gram per minute, respectively.

Statistical analysis

Analysis of variance was carried out by using SPSS 11.5 (SPSS Inc., Chicago, IL, USA) for all sets of data, and significant differences between means were determined by Duncan’s test. Differences were considered as statistically significant at P < 0.05.

Results

Ca²⁺ flux changes

The Ca²⁺ flux of the tobacco samples with or without aphid infestation was measured for 340 s with an interval time of 20 s. Figure 1(a) shows the plot of the Ca²⁺ influx versus time for tobacco samples with different infestation treatment. The Ca²⁺ efflux for the CK sample was shown in Figure 1(a) as well. The Ca²⁺ influx of tobacco samples after aphid infestation was slightly changed with time, but it seems the basic tendency of Ca²⁺ influx not to be changed. At first nine measurement points, the Ca²⁺ influx for all three tobacco samples, which were infested by aphids for 2 h, 15 h, and 5 d, respectively, exhibited similar change tendency. However, the Ca²⁺ influx of the infested sample of 15 h was decreased with time since the 10th measurement point. The behavior of Ca²⁺ flux for CK sample was significantly different from the infested samples. For CK sample, the Ca²⁺ flux showed efflux and little change with time. The fact that there was not sudden alteration of flux between CK and infested samples even at the first point indicated the Ca²⁺ flux response would be happened at beginning of infestation.

Figure 1. Ca²⁺ flux changes in aphid-infested tobacco for 0 h (CK), 2 h, 15 h, and 5 d. (a) Transient Ca²⁺ flux responses of tobacco mesophyll cells with time. Bars represent the standard deviation; (b) Average Ca²⁺ fluxes within the measuring period. Columns labeled with different letters (a and b) denote significant difference at P < 0.05 by Duncan's test.

In order to better understand the effect of the infestation time on Ca²⁺ flux, the average Ca²⁺ flux of all samples in the period of measurement time was shown in Figure 1(b). The average values of Ca²⁺ flux were 27.27, −31.52, −24.99, and −26.77 pmol·cm⁻²·s⁻¹, for samples infested by aphids for 0 h, 2 h, 15 h, and 5 d, respectively. Slight difference was observed for the infested samples with different infestation time. The infested sample of 2 h exhibited the highest Ca²⁺ influx and the sample of 15 h had the lowest Ca²⁺ influx, but similar with the value for the sample of 5 d, suggesting the Ca²⁺ influx was close to reach a constant value for tobacco samples after infestation for 15 h. Significant Ca²⁺ efflux was also found in the control tobacco (F = 12.35, df = 3, P < 0.05).

H₂O₂ flux changes

At the same infestation conditions as Ca²⁺ flux measurement, H₂O₂ flux of all samples, including CK and the infested samples, was measured. It was found that H₂O₂ flux exhibited influx phenomenon for the CK sample, as shown in Figure 2(a), which shows plots of H₂O₂ flux versus time for tobacco samples with different infestation treatment of 0 h, 2 h, 15 h, and 5d. However, after aphid infestation, H₂O₂ was considerably generated in tobacco mesophyll cells and exhibited continuous efflux, as shown in Figure 2(a). The H₂O₂ efflux of the samples infested by aphids for 2 h and 5 d showed a flat tendency though there were wave changes among measurement points. For the sample infested for 15 h, the H₂O₂ efflux seems to be slightly increased with increasing measurement time, especially after the 10th point.

Figure 2. H₂O₂ flux changes in aphid-infested tobacco for 0 h (CK), 2 h, 15 h, and 5 d. (a) H₂O₂ flux responses of tobacco mesophyll cells with time. Bars represent the standard deviation; (b) Average H₂O₂ fluxes within the measuring period. Columns labeled with different letters (a and b) denote significant difference at P < 0.05 by Duncan’s test.

The average H₂O₂ flux of all samples in the period of measurement time was shown in Figure 2(b). The average values of H₂O₂ flux were −0.35, 0.31, 0.73, and 0.35 pmol·cm⁻²·s⁻¹, for samples infested by aphids for 0 h, 2 h, 15 h, and 5 d, respectively. It was seen that the H₂O₂ efflux of the sample after aphid infestation for 15 h was the strongest.

POD and CAT activities

POD antioxidant enzyme activities for all aphid-infested tobacco samples were significantly higher than that of the CK samples (F = 46.57, df = 5, P < 0.05), as shown in Figure 3(a). The three infested samples exhibited similar POD antioxidant enzyme activity of around 700 μ·g⁻¹·min⁻¹ but the sample infested for 3 d had a slight higher POD antioxidant enzyme activity. It indicated that the extent of infestation by aphids for tobaccos did not influence the POD antioxidant enzyme activity. The POD antioxidant enzyme activity of CK samples was unexpectedly changed with time. The CK sample measured on the first day showed the highest POD antioxidant enzyme activity. Then, the POD antioxidant enzyme activities were decreased for the CK samples of 2 d and 3 d.

Figure 3. (a) POD activities in aphid-infested tobacco for 0 d, 1 d, 2 d, and 3 d; (b) CAT activities in aphid-infested tobacco for 0 d, 1 d, 2 d, and 3 d. Columns labeled with different letters (a, b, and c) denote significant difference at P < 0.05 by Duncan’s test.

The CAT antioxidant enzyme activities for the CK samples measured on the second and third day were slightly higher than that measured on the first day, as shown in Figure 3(b). The CAT antioxidant enzyme activity of the infested sample for 1 d was not removed much by comparison with the CK samples. However, the CAT antioxidant enzyme activities for the tobacco samples, which were infested by aphids for two and three days were significantly increased (F = 8.79, df = 5, P < 0.05), close to 2.5 times to the sample infested for one day. It indicated that the infestation of tobacco aphids resulted in a change in defense response.

Discussion

The role of ion flux in triggering and regulating defense responses has been widely discussed in most of previous studies (Lecourieux et al. 2006; Nemchinov et al. 2008; Hu et al. 2009; Wu & Baldwin 2009; Shabala et al. 2011). Ca²⁺ ion, as an important second messenger, is able to transport and amplify signals originating from cellular receptors and then stimulate the defense response in host plants (Garcia-Brugger et al. 2006). The cytosolic Ca²⁺ concentration could be of a rapid elevation for the plant defense responses in transduction pathways induced by the insect-specific elicitors. The data of Ca²⁺ flux, which was immediately changed from efflux for CK to influx for the infested tobacco samples, in this work are consistent with those previous reports (Maffei et al. 2006; Howe & Jander 2008). Nemchinov et al. (2008) found that Ca²⁺ influx occurred only at the first stage of pathogen–host interaction of 0–7 h after infestation. The initial calcium uptake was subsequently followed by the net calcium efflux initiated between 10 and 12 h and continued up to 48 h after infestation. However, in this work, the net calcium influx was neither significantly increased with infestation time, nor changed to the net calcium efflux. Will et al. (2007) studied molecular sabotage of plant defense by aphid saliva and addressed that sheath saliva prevents the calcium influx and that the watery aphid saliva appears to act as a calcium scaverger. The availability of calcium may also be reduced in the lumen of sieve elements (SEs) because of sealing of the puncture site in the SE cell wall. Thermodynamically, the passive Ca²⁺ efflux from the cell cytosol is impossible. Thus, Nemchinov et al. (2008) believed that PM Ca²⁺-ATPase was formed and pumped Ca²⁺ out because of an expanded cell death. In the insect–host interaction in this work, the tobacco mesophyll cells could be active as long as 5 d and the Ca²⁺ influx would not be significantly changed. This phenomenon was different from the pathogen–host interaction, in which pathogen would cause cell death in short infestation time. The Ca²⁺ flux was mostly dependent on the ion pathways on cytoplasmic membrane, rather than ion pump. Aphids can puncture all types of cells and readily seal almost every cell along the stylet pathway (Tjallingii & Esch 1993). In the stylet puncture, the components of the sheath and watery saliva of aphids play a pivotal role in establishing the physical and chemical constraints on the rise of calcium (Will & van Bel 2006). The phenomenon observed in this work should be further studied in the future.

ROS are generated during plant metabolism and play an important role in signal transduction and regulation (Shabala & Bose 2012). H₂O₂ is one of the most important ROS species involving in the signaling pathways to induce plant defense reactions against aphids (Martinez et al. 2003). H₂O₂ can be transported in long distances across cell membranes from its generation site. The previous study showed that Orgyia ericae-induced H₂O₂ accumulation in Ammopiptanthus mongolicus cells depended on Ca²⁺ influx, which representing an upstream signal affecting H₂O₂ accumulation (Gao et al. 2012). The phenomenon of H₂O₂ flux in this work is in agreement with the result in this reference. Ca²⁺ flux exhibited a strong influx at all infestation periods by aphids, while H₂O₂ showed an efflux behavior. The slight variation tendency of Ca²⁺ influx and H₂O₂ efflux was consistent.

The POD antioxidant activity of CK samples was observed to be changed. It might be related to the growth stage and the physiological status of tobacco. The similar phenomenon for host plants was also observed in literatures (Mao et al. 2011, 2012). After infestation, the POD antioxidant enzyme activity was significantly increased. POD and CAT antioxidant enzymatic systems in plants are believed to be disturbed, to some extents, by insect piercing-sucking (Han et al. 2009). After disturbance, the balance of the host plant metabolism could be resumed by POD antioxidant enzyme working with superoxide dismutase and CAT antioxidant enzyme through scavenging surplus free radicals. The role of CAT antioxidant enzyme is to scavenge the H₂O₂ produced in photorespiration and mitochondrial electron transport. The total nonspecific POD and CAT antioxidant enzyme activities were increased at all infestation time as a result of aphid feeding, suggesting the metabolism balance was broken by aphid feeding and a new balance was built.

Will and van Bel (2006, 2008) and Will et al. (2013) proposed that the Ca²⁺ influx could be a consequence of the mechanical damage and stretch-activated calcium-channels instead of the aphid saliva, in which contains calcium-binding proteins. Aphids can ingest phloem sap continuously for many hours or even days from a single SE, indicating that the gelling saliva protect the stylets from the plant response defense after the Ca²⁺ signal and H₂O₂ responses. Based on the results in this work that Ca²⁺ signal and H₂O₂ responses were not decreased but rather were slight increased after the stylet penetration, the defense response seems not to be ceased even under the prevention of sheath saliva sealing effect from the influx of Ca²⁺ (Will et al. 2007). The chemical compounds in aphid saliva would induce signaling pathways of salicylic acid and jasmonic acid (Will et al. 2013). During those reactions, the relative enzyme activities would be enhanced and respond to the insect attacks. In a long period of aphid feeding, the Ca²⁺ influx and H₂O₂ efflux were increased. POD and CAT antioxidant enzyme activities were significantly enhanced.

Acknowledgments

Thanks are given to Dr W.J. Wang for his technical assistance in the NMT testing.

Funding

This work was financially supported by the Ministry of Agriculture of China [grant number 201203091].

Additional information

Funding

Funding: This work was financially supported by the Ministry of Agriculture of China [grant number 201203091].