Reactive Oxygen Species, Oxidative Damage and Their Production, Detection in Common Bean (Phaseolus vulgaris L.) under Water Stress Conditions

Abstract

Reactive oxygen species (ROS) being small and highly reactive oxygen containing molecules play significant role in intracellular signaling and regulation. Various environmental stresses lead to excessive production of ROS causing progressive oxidative damage and ultimately cell death. This increased ROS production is, however, tightly controlled by a versatile and cooperative antioxidant system that modulates intracellular ROS concentration and controls the cell’s redox status. Furthermore, ROS enhancement under stress serves as an alarm signal, triggering acclimatory/defense responses via specific signal transduction pathways involving H2O2 as a secondary messenger. Nevertheless, if water stress is prolonged over to a certain extent, ROS production will overwhelm the scavenging action of the anti-oxidant system resulting in extensive cellular damage and death. DAB (3,3′-diaminobenzidine) test serves as an effective assessment of oxidative damage under stress. It clearly differentiates the lines on the basis of darker staining of leaves under water stress. The lines showing greater per cent reduction in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets.

1. Introduction

Abiotic stresses such as drought and high temperature invariably cause unfavorable changes in water status of plant cells as well as evolution of reactive oxygen species in cellular compartments resulting in acceleration of leaf senescence through lipid peroxidation and other oxidative damage [1]. Omae et al. [2] discovered a link between genotypic differences in bean leaf water status and crop productivity under drought conditions. This implies that there are differences in leaf water status among bean cultivars, which could be related to drought tolerance mechanisms. Among the reactive oxygen species hydrogen peroxide (H₂O₂) is a non-radical reactive oxygen species (ROS) produced in a two- electron reduction of molecular oxygen. H₂O₂ being a strong oxidant, it can initiate localized oxidative damage in leaf cells leading to disruption of metabolic function and loss of cellular integrity, actions that result in senescence promotion.

In plants, reactive oxygen species (ROS) are formed by the leakage of electrons from the electron transport activities of mitochondria, chloroplasts and plasma membranes or as a byproduct of various biotic and abiotic stresses due to disruption of cellular homeostasis [3, 4, 5]. A cell is said to be under oxidative stress when the level of ROS exceeds the defense mechanism. Increased ROS production during various stresses endangers cells, causing lipid peroxidation, protein oxidation, enzyme inhibition, nucleic acid damage, activation of the programmed cell death pathway, and ultimately cell death [6, 7, 8]. The overproduction of H₂O₂ has been observed in plants exposed to a number of stress conditions and is considered as one of the factors causing oxidative stress [9].

2. ROS, sites of production and their effects

Reactive oxygen species are a group of free radicals, reactive molecules and ions that are derived from o_2. ROS are known for playing role as both deleterious and beneficial species depending on their concentration in plants. They are produced at several locations within the cell in both stressed and unstressed cells (Figure 1).

Figure 1.

Sites of production of reactive oxygen species (ROS) in plants.

Production and removal of ROS needs to be controlled to avoid oxidative stress. When this level exceeds the defense mechanisms, a cell is said to be in a state of “oxidative stress”. Increased level of ROS can cause damage to biomolecules like lipids, proteins and DNA (Figure 2). These reactions can alter intrinsic membrane properties like fluidity, loss of enzyme activity, ion transport, protein cross-linking, DNA damage, inhibition of protein synthesis ultimately resulting in cell death.

Figure 2.

Reactive oxygen species (ROS) induces oxidative damage to lipids, proteins and DNA.

Under water stress, ROS production is enhanced in various ways. Inhibition of carbon dioxide assimilation coupled with changes in photosystem activities and photosynthetic transport capacity results in increased production of ROS [10]. Excess light energy dissipation in the PSII core and antenna generates ROS, which are potentially dangerous under water stress conditions [11]. The photorespiratory pathway is also increased, especially when RUBP oxygenation is optimum due to CO2 fixation limitation.

DAB (3,3′-diaminobenzidine) assay has been suggested as an effective qualitative assessment of plant response to biotic and abiotic stress and measures the intensity of oxidative burst under stress. Since the oxidative burst is an early response to stress, in terms of production of reactive oxygen species (ROS) including hydrogen peroxide through either NADPH oxidases or peroxidises (Bindschedler et al., 2006) that may exist singly or in combination in different plant species have been proposed for the generation of ROS. The qualitative evolution can be differentially tracked in different parts of plant under stress to assess the most vulnerable part under stress. It is done by staining with 3,3′-diaminobenzidine (DAB) which is oxidized by hydrogen peroxide and generates a dark brown precipitate.

3. Experimental method

The present study was conducted during 2016-2018 at the Division of Genetics & Plant Breeding, Faculty of Agriculture Wadura, SKUAST-K, Sopore. In the current study, fifty genotypes of common bean were evaluated under controlled conditions. The genotypes used were chosen based on their yield screening trial performance and represented a wide range of market classes in terms of use category, growth habits, and seed characteristics. The material included 47 breeding lines as well as three released varieties, SR-1, SFB-1, and Arka Anoop. The experiment was designed in a completely randomized design.

The DAB assay was performed in accordance with Daudi & O’Brien [12]. In this protocol, hydrogen peroxide (one of several reactive oxygen species) is detected in situ by staining with 3,3′-diaminobenzidine (DAB). In the presence of some haeme-containing proteins, such as peroxidases, DAB is oxidized by hydrogen peroxide to produce a dark brown precipitate. This precipitate is used as a stain in plant cells to detect the presence and distribution of hydrogen peroxide. DAB staining solution was prepared by adding 50 mg DAB and 45 ml sterile H₂O for a final 1 mg ml⁻¹ DAB solution in a 50 ml falcon tube. The tube was covered with aluminum foil as DAB is light-sensitive. About 25 μl Tween 20 (0.05% v/v) and 2.5 ml 200 mM Na₂HPO₄ to the DAB solution to produce 10 mM Na₂HPO₄ DAB staining solution. Similar, fully opened leaves were selected from each treatment and incubated for one hour in falcon tubes with 2 ml of the DAB staining solution with the volume being adjusted to ensure that leaves were immersed. The leaves from irrigated treatment were incubated with 2 ml of 10 mM Na₂HPO₄. All the falcon tubes from both drought and irrigated treatments were shaken for 4-5 h at 80-100 rpm. Following the incubation, the aluminum foil was replaced and the DAB staining solution replaced with bleaching solution (ethanol: acetic acid: glycerol in ratio of 3:1:1). For 15 minutes, the falcon tubes were immersed in a boiling water bath (90-95°C). The chlorophyll will be bleached out, but the brown precipitate formed by the DAB reacting with the hydrogen peroxide will remain. The time should be adjusted (5 minutes) depending on how the leaves look (they should be completely devoid of chlorophyll). After 15 minutes of boiling, the bleaching solution was replaced with fresh bleaching solution and left to stand for 30 minutes. DAB staining was visualized directly on leaves.

4. Results and discussion

The DAB staining of common bean genotypes under irrigated and water stress conditions clearly differentiates the lines on the basis of darker staining of leaves under drought (Figure 3). The lines showing greater per cent reductions in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets. However, DAB is only a qualitative test for evolution of reactive oxygen species such as H₂O₂ and the genotypes showing greater staining under drought can be further analyzed for the amount of H₂O₂ through various analytical methods.

Figure 3.

DAB staining of common bean (Phaseolus vulgaris L.) genotypes under water stress conditions. Largely stained genotypes (3b) show higher production of hydrogen peroxide under stress conditions causing oxidative damage to cell structure.

The DAB staining of common bean genotypes clearly differentiates the genotypes on the basis of darker staining of leaves under water stress. The lines showing greater per cent reductions in yield parameters show greater staining in DAB assay underlining the reliability of using this assay as a reliable supplement to phenotyping protocols for characterizing large germplasm sets (Table 1). Reactive oxygen species play an important role as signaling molecules that initiate stress responses in plants. Environmental stresses are known to induce the production of H₂O₂ and other toxic oxygen species in cellular compartments, resulting in the acceleration of leaf senescence via lipid peroxidation and other oxidative damage, according to Upadhyaya et al. [1]. Because H₂O₂ is a strong oxidant, it can cause localized oxidative damage in leaf cells, disrupting metabolic function and causing cellular integrity loss, both of which promote senescence. Overproduction of H₂O₂ has been observed in plants subjected to a variety of stress conditions and is thought to be one of the causes of oxidative stress [9]. According to Foyer and Noctor [13], among the various forms of ROS, the central role in plant signaling, regulating plant development, and adaptation to abiotic and biotic stresses is played by hydroxyl radicals. ROS also act as signaling molecules to regulate development and stress responses [14]. Increased availability of H₂O₂ is commonly observed feature of plant stress response signature. The physiological context involves a continuous supply of environmental stimuli that can trigger intracellular H₂O₂ accumulation or modulate the response to such accumulation.

Genotype	Pods per plant			Pod length (cm)			Seeds per pod			100-seed weight (g)			Seed yield per plant (g)
	S	NS	PC	S	NS	PC	S	NS	PC	S	NS	PC	S	NS	PC
WB-6	7.22	12.77	−43.48	10.73	13.71	−21.76	3.28	3.81	−14.02	39.34	47.66	−17.45	8.78	19.55	−55.09
WB-22	8.64	14.11	−38.76	9.63	12.14	−20.67	3.86	4.26	−9.27	44.39	50.57	−12.21	14.35	29.32	−51.04
WB-83	14.33	23.74	−39.62	8.60	11.11	−22.58	4.51	4.95	−8.98	24.72	28.20	−12.34	16.33	24.67	−33.81
WB-112	10.35	15.86	−34.74	11.78	13.08	−9.94	3.49	4.09	−14.65	41.27	44.94	−8.16	14.97	26.06	−42.54
WB-185	14.34	20.24	−29.14	11.90	12.98	−8.35	3.61	4.52	−20.22	40.31	44.36	−9.14	21.74	34.44	−36.88
WB-216	7.07	10.46	−32.39	11.02	13.96	−21.08	3.19	3.68	−13.29	40.93	47.22	−13.32	12.17	16.28	−25.27
WB-222	18.82	22.13	−14.95	9.10	10.09	−9.80	4.27	4.60	−7.27	24.99	28.28	−11.61	21.23	31.10	−31.73
WB-257	10.70	15.33	−30.09	11.30	12.97	−12.91	3.24	3.93	−17.43	41.87	48.72	−14.05	17.41	25.38	−31.41
WB-341	21.06	23.05	−8.61	9.61	10.47	−8.17	4.50	4.98	−9.64	26.68	29.00	−8.00	27.24	33.18	−17.88
WB-401	14.57	17.97	−18.91	9.06	10.36	−12.55	3.76	4.14	−9.18	25.36	27.65	−8.29	11.68	15.10	−22.61
WB-451	17.00	27.28	−37.67	8.47	10.35	−18.20	3.63	4.14	−12.30	25.98	29.57	−12.12	23.17	33.72	−31.28
WB-956	9.55	15.55	−38.57	11.56	12.93	−10.59	3.68	4.57	−19.47	38.21	42.84	−10.80	18.61	25.41	−26.74
WB-1446	11.47	14.11	−18.74	10.37	13.23	−21.62	3.52	4.06	−13.30	35.31	39.03	−9.53	17.84	24.96	−28.53
WB-1492	8.22	12.39	−33.68	9.60	10.84	−11.39	3.61	4.05	−10.97	29.42	32.13	−8.43	13.16	27.35	−51.86
WB-1587	4.16	9.07	−54.13	7.34	9.96	−26.34	3.12	3.77	−17.35	25.17	28.95	−13.07	8.26	13.50	−38.77
WB-1634	21.22	24.90	−14.77	10.62	11.67	−8.99	4.74	5.16	−8.13	25.18	26.71	−5.74	31.96	36.05	−11.33
WB-1643	16.05	19.27	−16.70	11.96	13.11	−8.77	3.79	4.61	−17.68	27.73	31.94	−13.18	20.69	25.52	−18.90
SR-1	6.84	10.61	−35.56	8.20	14.79	−44.52	3.32	4.44	−25.31	35.27	39.51	−10.73	13.43	20.18	−33.44
SFB-1	11.72	25.55	−54.12	13.92	16.90	−17.65	3.91	4.83	−19.13	23.15	26.53	−12.75	18.57	22.11	−16.01
Arka Anoop	10.71	17.17	−37.61	−12.73	15.78	−19.35	3.23	4.07	−20.64	24.00	29.60	−18.93	12.50	17.69	−29.33
Mean	12.26	17.73	30.88	10.38	12.52	17.09	3.71	4.34	14.52	31.97	36.17	11.61	17.21	25.08	31.38
C.D (p ≤ 0.05)	Genotypes = 2.882; Water regime = 0.911; G x WR = 4.075			Genotypes = 0.952; Water regime = 0.301; G x WR = 1.346			Genotypes = 0.355; Water regime = 0.112; G x WR = 0.247			Genotypes = 1.572; Water regime = 0.497; G x WR = 2.223			Genotypes = 3.069; Water regime = 0.979; G x WR = 4.379

Table 1.

Mean performance under different water regimes and percent reductions for yield and yield parameters under water stress in common bean (Phaseolus vulgaris L.).

The detection of cellular levels of H₂O₂ was done by DAB staining method and our results shows a clear difference in the degree of staining achieved in the stressed plant. Under water stress, there was significant variation in staining in different genotypes indicating differential oxidative damage on account of production of H₂O₂. The lines which showed fair amount of tolerance to water stress in terms of higher yield and lower reduction had almost negligible staining while as the genotypes which showed lower yield showed higher reduction, distinctly darker staining. Less tolerant cultivars accumulated more H₂O₂ than more tolerant ones, and vulnerable variety showed noticeably greater increases in lipid peroxidation. Similar findings were reported by a number of prior studies (Chai et al., 2005; Zlatev et al., 2006).

Plants accumulate reactive oxygen species during drought stress (Verslues et al., 2006). ROS can cause cell death by destroying DNA, proteins, and carbohydrates through partially reduced or activated oxygen derivatives [3]. DAB staining investigations can efficiently differentiate ROS levels in transgenic lines of rice, with reduced staining in transgenic lines compared to control plants after drought-stress treatment, according to Jiang et al. (2016). DAB assay results were consistent with those obtained using membrane stability and other biochemical parameters in tolerant and sensitive wheat cultivars, according to Chakraborty and Pradhan (2012). However, only a qualitative assessment of DAB was performed in this work. Ghahfarokhi et al. (2016) performed an experiment to examine the effects of drought stress caused by withholding irrigation at the vegetative stage (4-5 leaves) and reproductive stage on crop production, physiological, and biochemical features in hybrids of maize (Zea mays L.) (anthesis). Results indicated that these traits were significantly impacted by drought stress (Table 2). Under water stress, both the yield and its constituent parts significantly reduced. The main causes of the yield reduction were a decrease in the quantity of grain ear⁻¹ and the weight of 1000 grains. In comparison to other hybrids, short maturity hybrids had a larger yield reduction. These results suggested that water stress lead to the production of reactive oxygen species (ROS), which caused an increased membrane permeability and oxidative stress in the maize plants. The reliability of the DAB test can be further validated by conducting a quantitative assessment of hydrogen peroxide evolution in common bean under water stress.

Parameter	Common bean
	Susceptible lines		Tolerant lines
	WB-285575	WB-335	WB-216	WB-451
Canopy temperature depression (°C)	−1.07	−1.84	−0.68	−2.58
Relative water content (%)	18.82	9.70	10.69	11.54
Membrane stability index	0.456	0.592	0.459	0.656
Chlorophyll stability index	0.675	0.336	0.295	0.174
Proline content (μmol/g)	20.65	22.63	36.72	37.33

Table 2.

Comparative performance of tolerant susceptible cultivars for various physio- biochemical parameters under stress in common bean.

5. Conclusion

ROS are produced by electron transport activities of mitochondria, chloroplast, plasma membrane or as a byproduct of various metabolic pathways localized in different cellular compartments. Study of formation and fate of ROS using advanced and analytical techniques help in developing broader view of the role of ROS in plants. DAB assay is employed to delineate genotypic response in terms of qualitative differentiation of oxidative damage as indicated by differential staining under DAB treatment. All the genotypes revealed almost similar staining in irrigated conditions. While as, under drought conditions, genotypes which showed better resilience to water stress in terms of higher yield and drought had significantly lesser staining as compared to susceptible ones. Therefore, DAB staining can be used as complementary method for differentiating genotypes to water stress.