Real-time monitoring of superoxide anion radical generation in response to wounding: electrochemical study

Introduction

The formation of reactive oxygen species (ROS) in plants is an unavoidable consequence of photosynthesis (Ledford, Chin & Niyogi, 2007; Alessandro et al., 2011; Foyer & Shigeoka, 2011; Laloi & Havaux, 2015). The introduction of molecular oxygen into the environment by photosynthetic organisms during the evolution of aerobic life is associated with the formation of ROS (Tripathy & Oelmüller, 2012). Plants growing in a fluctuating environment are exposed to various biotic stresses such as bacteria, viruses, fungi, parasites, insects, weeds, etc. and abiotic stresses such as fluctuations in temperature, salinity, water, radiation, toxic chemicals and mechanical stress which are closely linked to higher ROS production. The chloroplasts, mitochondria, and peroxisomes are among the chief organelles involved (Elstner, 1991; Foyer & Harbinson, 1994; Asada, 1996; Turrens, 2003; Liu et al., 2007; Murphy, 2009). As a response, ROS, including the superoxide anion radical (O₂^•−), hydroperoxyl radical (HO₂^•), hydrogen peroxide (H₂O₂), hydroxyl radical (HO^•), singlet oxygen (¹O₂), peroxyl radical (ROO^•), hydroperoxide (ROOH) and alkoxyl radical (RO^•), are produced (Miller, Shulaev & Mittler, 2008; Gill & Tuteja, 2010; Foyer & Noctor, 2005; Asada, 2006; Miller et al., 2009; Bhattacharjee, 2010; Choudhury et al., 2013).

The production of ROS by an oxidative burst is an imperative element of the wound response in algae, plants, and animals (McDowell et al., 2015). As a response to wounding, plants release oligosaccharide cell wall fragments, which play an important role in the signaling cascade that initiates an intense, localized production of ROS (Legendre et al., 1993; John et al., 1997; Stennis et al., 1998). Wounding stimulates the production of O₂^•−, H₂O₂ and nitric oxide (NO), which can directly attack encroaching pathogens at the site of the wound (Murphy, Asard & Cross, 1998; Garces, Durzan & Pedroso, 2001; Jih, Chen & Jeng, 2003). In Arabidopsis thaliana leaves measured under ambient light conditions, O₂^•− and H₂O₂ mainly originate from photosynthetic electron transport, predominantly at the site of wounding (Morker & Roberts, 2011). The role of NADPH oxidase in ROS production, however, was not completely ruled out. Therefore, the generation of O₂^•− and H₂O₂ can be attributed to the collective effect of wounding and light stress. O₂^•− generation in the root cells of plants in response to wounding has been studied by electron paramagnetic resonance (EPR) spin-trap spectroscopy and epinephrine-adrenochrome acceptor methods (Vylegzhaninat et al., 2001). Tiron (4, 5-dihydroxy-1, 3-benzene-disulfonic acid disodium salt) was used, and the tiron semiquinone EPR spectra showed O₂^•− generation. The level of O₂^•− production in the roots was measured with epinephrine which in the presence of O₂^•− is converted to adrenochrome and can be monitored at 480 nm by a spectrophotometer (Misra & Fridovich, 1972; Barber & Kay, 1996). In addition to spectroscopy, staining with dye, such as tetrazolium dye and nitro blue tetrazolium (NBT), has been used to detect the production of O₂^•− in situ, with visualization of O₂^•− generation as a purple formazan deposit within leaflet tissues (Wohlgemuth et al., 2002).

Although various methods, such as EPR spin-trapping spectroscopy (Von, Schlosser & Neubacher, 1993), chemiluminescence (Anderson et al., 1991), the reduction of NBT and the reduction of the redox protein cytochrome c (Doke, 1983b; Doke, 1983a; Doke, 1985) have been used to detect and monitor O₂^•−, each of these methods has inadequate specificity and sensitivity. EPR spin-trapping spectroscopy is one of the most sensitive and specific method for ROS detection; however, kinetic measurements are not possible at the current stage of development. Chemiluminescence, also known as ultra-weak photon emission, has been widely used recently as a non-invasive method to understand the involvement of ROS in oxidative radical reactions (Prasad & Pospíšil, 2012; Prasad & Pospíšil, 2013; Pospíšil, Prasad & Rác, 2014); however, limitations with respect to the specificity for particular ROS involvement exists (Halliwell & Gutteridge, 1989).

Integration of metalloporphyrins into electropolymerized polymer electrodes have been developed rigorously over the last years because these materials are effective electrocatalysts for chemical as well as photochemical applications (Bedioui et al., 1995). Numerous authors have recently tested the potential use of electropolymerized metalloporphyrins as new electrode materials for chemical and biological sensors (Deronzier & Moutet, 1996; Yim et al., 1993; Bedioui, Trevin & Devynck, 1996). In our current study, we provide an experimental approach for the detection of O₂^•− by polymeric iron-porphyrin-based modified carbon electrode based on the reaction mechanism presented in Fig. 1A (Yuasa & Oyaizu, 2005). Detection of O₂^•− by highly sensitive and selective polymeric iron-porphyrin-based modified carbon electrodes was tested in in vivo leaf sample subjected to wounding. The current study introduces the use of catalytic amperometric biosensors for the real-time detection of O₂^•−.

Material and Methods

Experimental conditions for real-time monitoring of the oxidation current of O₂^•−

The electrochemical detection of O₂^•− was measured using the X/XO system based on the method described in our recent study (Matsuoka et al., 2014) (Fig. 2). The subsequent oxidation current for O₂^•− was monitored using polymeric iron-porphyrin-based modified carbon electrodes (φ = 1 mm) with Ag/AgCl as the reference electrode.

Spinach leaves were fixed on a petri-dish with a diameter of 60 mm using double-sided adhesive tape. A total of 10 mM phosphate buffer saline (pH 7.2) (PBS) was gradually added to maintain a sufficient volume to submerge the whole spinach leaf in PBS. During the measurement, injury was performed using a glass capillary with an inner diameter of about 1.2 mm and wall thickness of 200 µm as presented in Fig. 1B and Data S1. For data presented in the manuscript, the injury/wounding in spinach leaves were done either one time or multiple times (between 8–10 times) while to visualize the state of leaves, injury/wounding was made one time, five times and 20 times (Data S1). Mechanical injury and mechanical wounding were performed close to the site of the electrode. The oxidation current was measured at +0.5 V vs. Ag/AgCl at room temperature.

Results

Real-time monitoring of O₂^•− generation during wounding of spinach leaves

To validate that there is no interference in the measurement caused by the suspension of spinach leaf in PBS, the oxidation current was measured in a non-wounded spinach leaf suspended in PBS (Fig. 3). No fluctuation in the oxidation current of O₂^•− was observed in the non-wounded spinach leaf suspended in PBS, (Fig. 3), whereas a negligible fluctuation was observed with the exogenous addition of SOD (data not shown). These results indicate that these chemical species (PBS and SOD) do not interfere with the measurements. The kinetics of the production of O₂^•− were also measured in the chemical system containing no spinach leaves and in the presence of SOD (400 U ml⁻¹) indicating no significant fluctuation in oxidation current (Data S4).

Real-time monitoring of the oxidation current for O₂^•− was performed in spinach leaves where mechanical injury was stimulated one time using a glass tube (Fig. 4A) and mechanical wounding was done multiple times (Fig. 4B). The wounding in spinach leaves was done one time (4A) and multiple times (between 8–10 times) (4B) using a glass capillary with an inner diameter of about 1.2 mm and wall thickness of 200 µm. The results indicate that the O₂^•− production increased considerably with the dose of mechanical injury (Fig. 4). Furthermore, to visualize the extend of damage to leaf during mechanical injury induced by glass capillary, photograph of leaves showing the physiological state have been presented along with kinetics on real-time monitoring of the oxidation current of O₂^•− under experimental condition mentioned in dataset presented (Data S1). To determine the concentration of O₂^•− generated in mechanically injured spinach leaves, the calibration curve was established for various concentrations obtained using standard X/XO system (Fig. 2). A maximum oxidation current (Δi) of 1.5 nA (at time span, 60 s) and 7.5 nA (at time span, 300 s) was observed in mechanically injury made at a minimal dose (Fig. 4A) and at multiple sites (Fig. 4B). Based on the data obtained and the maximum oxidation current recorded, the O₂^•− was calculated and expected production was found to be about 40 nM (4A) and about 200 nM (4B) (Table 1).

Table 1:

Calculation.

Superoxide anion radical (O₂^•− concentration calculated using standard calibration curve (R² = 0.9918) (Fig. 2). The total change in oxidation current was found to be 1.5 nA (Δi) for minimal dose of injury (Fig. 4A) and 7.5 nA (Δi) for injury at multiple sites (Fig. 4B). The total O₂^•− concentration was found to be equivalent to 40 nM (Fig. 4A) and 200 nM (Fig. 4B) at 60 s and 300 s, respectively.

	A	B
Δi (nA)	1.5	7.5
Δt (s)	60	300
O2•− (nM)	40	200

DOI: 10.7717/peerj.3050/table-1

In addition, O₂^•− generation was also measured in spinach leaves under the effect of wounding at room temperature in presence of exogenous addition of SOD (Fig. 5). In the absence of wounding, as observed during the first minute of real-time monitoring, no considerable change in the oxidation currents of O₂^•− was observed. However, wounding instantaneously resulted in a fast increase in the oxidation current for O₂^•− of approximately 10 nA, followed by a gradual decrease, which continued for more than 10 min. To confirm the production of O₂^•−, the effect of SOD, which leads to the dismutation of O₂^•− to H₂O₂, on the oxidation current in a wounded spinach leaf was analyzed. The addition of 400 U ml⁻¹ SOD suppressed the oxidation current for O₂^•− from 3.5 nA to 2 nA (Fig. 5). However, complete suppression of the oxidation current was not observed. The oxidation current, which persisted at approximately 1 nA for a few minutes, can be attributed to rapid O₂^•− diffusion to the electrode before its conversion to H₂O₂ or to the limited SOD activity at a fixed concentration. The effect of SOD (400 U ml⁻¹) added exogenously was also measured at the point of maximum oxidation current where a comparatively higher suppression was recorded (Fig. 6).

Discussion

In addition to plants, ROS detections have been performed in model system including animals. During recent past, Zuo and coworkers (2011, 2013) presented results on intracellular ROS formation in single isolated frog myofibers during low P_O2 conditions using dihydrofluorescein (Hfluor), a fluorescein analog of DCFH. Cyt c assay was also used to measure O₂^•− in contracting skeletal muscle in pulmonary TNF- α overexpression mice (Zuo et al., 2014; Zuo et al., 2004). Several mechanisms for the generation of ROS involving O₂^•− have been suggested. It has been proposed previously that an NADPH oxidase-like enzyme in the plant plasma membrane is involved in the production of O₂^•− which is then converted to the more stable H₂O₂ during the oxidative burst in response to pathogen attack of plant cells (Murphy & Auh, 1996; Doke et al., 1996). In skeletal muscle, the major source of ROS especially extracellular O₂^•− formation is via the arachidonic acid metabolism through lipoxygenase (LOX) activity (Zuo et al., 2004). However, in contrast to the view that wound-induced ROS are primarily produced extracellularly by NADPH oxidase enzymes (Watanabe & Sakai, 1998; Flor-Henry et al., 2004), it has been recently indicated that wound-induced O₂^•− and H₂O₂ originate from photosynthetic electron transport measured at wounded sites under ambient light conditions (Morker & Roberts, 2011). The O₂^•− is produced during the electron transport process by the reduction of molecular oxygen in the chloroplasts and mitochondria. The authors proposed that O₂^•− and H₂O₂ production is linked to wounding, which is enhanced significantly under light conditions.

A recent report on Pisum sativum seedlings proposes a mechanism responsible for oxidative burst during wounding. During mechanical wounding, polyunsaturated fatty acids (PUFA), polyamines, LOX and peroxidases (Prx) are released in the extracellular matrix. Under these circumstances, LOX is involved in the oxidation of PUFA or polyamines, which induces diamine oxidases (DAO) to produce H₂O₂, further leading to O₂^•− production catalyzed by Prx (Roach et al., 2015). A diverse range of organisms use Prx to produce O₂^•−, e.g., Triticum sativum roots (Minibayeva et al., 2009), Castanea sativa and Trichilia seeds (Roach et al., 2009; Whitaker et al., 2010), liverworts (Li et al., 2010), and lichens (Liers et al., 2011). Another group of redox enzyme involved in the wound-induced oxidative burst is DAO (Rea et al., 2002; Cona et al., 2006; Yoda, Hiroi & Sano, 2006; Angelini et al., 2008), and cooperation between DAO and Prx is considered important in the wound response. In addition to DAO and Prx, LOX not only generates lipid hydroperoxides (LOOH) but can also generate O₂^•− via the oxidation of pyridine nucleotides and therefore considerably contributes to oxidative stress in cells (Roy et al., 1994). Lipid oxidation by LOX is an important part of the wound-response because oxylipins, the break-down products of lipid peroxides, can act as effective signaling molecules to rapidly induce transcriptional changes during wounding (Upchurch, 2008; Higdon et al., 2012). Plasma membrane-bound Prx can utilize PUFA such as linoleic acid released at the wound site for the synthesis of O₂^•−. Even if LOX is not known to be directly involved in O₂^•− production, it was suggested that extracellular LOX may play an important role by competing with Prx for fatty acids and producing reactive electrophiles that coordinate signaling responses (Doke et al., 1996).

The electrochemical method with the employment of different modified electrodes have been successfully standardized and applied for detection of varied ROS including O₂^•− during the recent past (Groenendaal Jonas et al., 2000; Yuasa & Oyaizu, 2005; Yuasa et al., 2005). The polymeric iron-porphyrin-based modified carbon electrode is a useful tool and provides a direct method for real-time monitoring and precise detection of O₂^•− in biological samples in-situ (Yuasa et al., 2005). The kinetic measurement showing the production of O₂^•− for a long range of time (minutes) presented in our study (https://ecs.confex.com/ecs/229/webprogram/Paper70675.html) have been demonstrated and thus opens a new area of investigation which have always been difficult to explore using other available methods such as EPR spin trapping spectroscopy, fluorescence microscopy and other biochemical methods etc. Thus, the electrode has strong potential for wide application in plant research for the specific and sensitive detection of O₂^•− and the kinetic behavior in real-time. In addition to points mentioned above, using synthetic porphyrin has an additional advantage. To date, O₂^•− sensors based on naturally derived enzyme (e.g., SOD, Cyt. c) have been developed (Di, Bi & Zhang, 2004). However, enzymes on the sensor are likely to be denatured. In contrast, the porphyrin based sensor can be used without denaturation. At the same time, the current method is cost effective. Certain limitations exist; the current polymeric iron-porphyrin-based modified carbon electrodes are light-sensitive, which might hinder its photo-electrochemical applicability (Brett & Brett, 1984).

Conclusion

In the current study, we present our polymeric iron-porphyrin based modified carbon electrode for application in real-time monitoring and precise detection of O₂^•− in biological system. It has strong potential for wide application in plant research for specific and sensitive detection of O₂^•−.

Supplemental Information