Elsevier

Plant Physiology and Biochemistry

Volume 154, September 2020, Pages 675-688
Plant Physiology and Biochemistry

Research article
Effects of ethylenediurea (EDU) on regulatory proteins in two maize (Zea mays L.) varieties under high tropospheric ozone phytotoxicity

https://doi.org/10.1016/j.plaphy.2020.05.037Get rights and content

Highlights

  • Role of regulatory proteins and enzymes were revealed by the protective mechanism of EDU in two maize varieties.

  • Defense and energy metabolism proteins showed optimum expression proving their role against high tropospheric O3.

  • EDU enhances antioxidant defense mechanism by enriching antioxidant content and regulating antioxidative enzymes.

  • The key factor inducing the varieties differences in EDU responses is that SHM3031 is sensitive and the PEHM5 is tolerant.

  • Our results are relevant for assessing the O3 risk to maizeand for including O3 affects in crop productivity models.

Abstract

Rising tropospheric ozone is a major threat to the crops in the present climate change scenario. To investigate the EDU induced changes in proteins, two varieties of maize, the SHM3031 and the PEHM5, (hereafter S and P respectively) were treated with three EDU applications (0= control, 50 and 200 ppm) (hereafter 0= A, 1 and 2 respectively) (SA, S1, S2, PA, P1, P2 cultivar X treatments). Data on the morpho-physiology, enzymatic activity, and protein expression (for the first time) were collected at the vegetative (V, 45 DAG) and flowering (F, 75 DAG) developmental stages. The tropospheric ozone was around 53 ppb enough to cause phytotoxic effects. Protective effects of EDU were recorded in morpho-physiologically and biochemically. SOD, CAT and APX together with GR performed better under EDU protection in SHM3031 variety than PEHM5. The protein expression patterns in SHM3031 at the vegetative stage (28% proteins were increased, 7% were decreased), and at the flowering stage (17% increased, 8% decreased) were found. In PEHM5, a 14% increase and an 18% decrease (vegetative stage) whereas a 16% increase and a 20% decrease (flowering stage) were recorded in protein expression. Some protein functional categories, for instance, photosynthesis, carbon metabolism, energy metabolism, and defense were influenced by EDU. Rubisco expression was increased in SHM3031 whereas differentially expressed in PEHM5. Germin like protein, APX, SOD, and harpin binding proteins have enhanced defense regulatory mechanisms under EDU treatment during prevailing high tropospheric O3. The present study showed EDU protective roles in C4 plants as proven in C3.

Introduction

Ground-level ozone (O3) is increasing at the rate of approximately 0.5–2% per year over the mid-latitudes of the Northern Hemisphere due to rapid industrialization and urbanization in the last three decades (IPCC, 2013; Simpson et al., 2014). Global tropospheric O3 levels were around 50 ppb in the year 2000, already 25% above the AOT40 threshold proven for damage to sensitive plants (Bhatia et al., 2012). Due to its phytotoxicity, tropospheric O3 has been recognized as one of the most hazardous and toxic air pollutants with a higher degree of negative impacts on global agriculture (Ashmore, 2005; Emberson et al., 2009; Singh et al., 2015). Various studies conducted on the Indian crops suggest their high vulnerability to ozone-induced damage, but unfortunately genetic variation among cultivars in response to O3 has hardly been addressed (Oksanen et al., 2013; Peng et al., 2020). Global yield reduction's, due to ambient O3, for maize, rice, wheat, and soybean have been estimated to be 6.1%, 4.4%, 7.1%, and 12.4% (mean of 2010–2012) annually, respectively (Mills et al., 2018). Economic losses for Europe based on ozone assessment studies on 23 crops, were estimated to be US$7.5 billion (Holland et al., 2006) and global crop production losses were estimated to have been 79–121 Mt worth US$11–18 billion (Avnery et al., 2011). Estimating the loss of crop production from ground-level O3 is valuable for understanding the potential benefits of reducing O3 concentration and for projecting future food supply (Burney and Ramanathan, 2014).

Among the different effects of ozone on vegetation, visible injury in leaves is considered a valuable tool for the assessment of ozone impacts in the field and the detection of areas of high risk due to O3 (Schaub et al., 2010). Ozone causes damage by entering the leaf intercellular air spaces via stomata, where it reacts with compounds in the exposed wet cell-wall surfaces, causing the production of damaging radicals and signaling that accelerates senescence (Long and Naidu, 2002; Fiscus et al., 2005). Photosynthetic efficiency and mesophyll conductance are also affected by ozone in the crops (Xu et al., 2019; Peng et al., 2020). This has led to the expectation that O3 damage will be less in C4 plants (maize and sugarcane), given their intrinsically lower stomatal conductance, as well as for plants under drought stress, and in response to rising (CO2) (McKee et al., 2000; Long and Naidu, 2002; Leitao et al., 2007; Yi et al., 2020). Different studies indicate that O3 damages the photosynthetic machinery leading to a progressive loss in the amount as well as activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (Agrawal et al., 2002; Cho et al., 2008). Light and dark reactions of chloroplast also get affected either directly or indirectly due to high ozone concentration (Fiscus et al., 2005).

Ethylenediurea (EDU) has been widely used as a research tool to reveal and evaluate the ozone-sensitivity in several crops and tree species (Paoletti et al., 2009; Feng et al., 2010; Manning et al., 2011; Oksanen et al., 2013). Protective capability of EDU was observed on reactive oxygen species (ROS) mechanism in wheat (Agrawal et al., 2005; Singh and Agrawal, 2009; Pandey et al., 2019), European Ash (Paoletti et al., 2008), mung bean (Singh et al., 2010a), carrot (Tiwari and Agrawal, 2010), maize (Singh et al., 2018) and in palak (Spinach) (Tiwari and Agrawal, 2009).

The present study comprises the evaluation of regulatory proteins together with morpho-physiological, biochemical, and yield in two maize varieties under EDU treatment. This is the first proteomic study under EDU treatment in the C4 crop. As we know, the morpho-physiological approach which reveals the changes in comparison with the given EDU treatments, whereas proteomic and biochemical response analyses the insight of the plant's metabolism under any prevailing conditions. Proteomic evaluation includes, identifying differential expression of proteins in response to EDU treatment in two maize varieties. The other study parameters include pigments estimation, lipid peroxidation (MDA equivalent content), antioxidants (Ascorbate and Glutathione), and the antioxidative enzymes: Ascorbate peroxidase (APX), Glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD). Maize is considered as the third most important crop at the global context of which, two varieties SHM3031 (stress sensitive), and PEHM5 (stress tolerant) were selected for the present study.

Section snippets

Study site, climatic condition and plant material

The study was conducted at CSIR-National Botanical Research Institute garden in Lucknow, city of Uttar Pradesh, India. It is situated along the southern bank of river Gomati at 26055′ N latitude, 80059 E longitude, and an altitude of 113 m in subtropical climates. Lucknow is characterized by a dry, tropical monsoon climate. Maximum average temperatures varied from 25 to 32 °C, and minimum average temperature varied from 14 to 27 °C, and a minimum of 60% and a maximum of 78% humidity was

Average ozone and AOT40

The average ambient ozone during the study period was 53.21 ppb. It had been recorded that precipitation and cloudy weather were responsible for comparatively low ambient ozone during the study period rather than dry seasons. Precipitation leads to washout of the precursor responsible for ozone formation. The ozone levels since October were higher than until September because of higher precipitation rate in later months (Fig. 1). High ambient O3 concentrations (hourly average) (>40 ppb)

Discussion

We demonstrated harmful effects of tropospheric ozone on maize using EDU as anti-ozonant supplement in the middle IGP region, of India. The higher concentration of ozone in troposphere resulted from high temperature, longer sunshine hours, and less relative humidity. In our study, the maximum O3 concentration (71.38 ppb) was found during October and November, and minimum during peak rainfall months (July, August, and September). The lower O3 concentration during rainy season was appeared due to

Conclusion

In Summary, under conditions with high tropospheric ozone as those prevailing in Lucknow area. EDU conferred an important protection to sensitive maize variety, increasing morpho-physiological performance, maintaining an enhanced antioxidant capacity, and finally leading to higher biomass and/or grain yields. The magnitude of variations in biochemical parameters varied with stages and varieties. SHM3031 showed more induction in non-enzymatic antioxidants with respect to PEHM5 at both the

Author's contribution

VP, and SKG designed the experiment. SKG, MS, and VKM did yield and physiological work. BM did the enzymatic work. SKG, MS,VKM, and FD did proteomic work. SKG, and VP analyzed the data and wrote the paper. SKG, ZJL, and VP revised the manuscript. All the authors approved the paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Authors are grateful to Director, CSIR-NBRI for providing necessary facilities. SKG and MS are grateful to CSIR and UGC, respectively for senior research fellowship. Funding for this work was provided by Council of Scientific & Industrial Research (CSIR), New Delhi, India [Project no. PSC112].

References (91)

  • S.K. Gupta et al.

    Impact of Ethylene diurea (EDU) on growth, yield and proteome of two winter wheat varieties under high ambient ozone phytotoxicity

    Chemosphere

    (2018)
  • R.L. Heath et al.

    Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation

    Arch. Biochem. Biophys.

    (1968)
  • K. Herbinger et al.

    Complex interactive effects of drought and ozone stress on the antioxidant defense systems of two wheat cultivars

    Plant Physiol. Biochem.

    (2002)
  • D. Le Thiec et al.

    Ozone and water deficit reduced growth of Aleppo pine seedlings

    Plant Physiol. Biochem.

    (2003)
  • Louis Leitao et al.

    Assessment of the impact of increasing concentrations of ozone on photosynthetic components of MAIZE (Zea mays L.), a C4 plant

    Environ. Pollut.

    (2007)
  • H.K. Lichtenthaler

    Chlorophylls and carotenoids: pigments of photosynthetic biomembranes

    Methods Enzymol.

    (1987)
  • S. Luo et al.

    Fe2+-catalyzed site-specific cleavage of the large subunit of ribulose 1, 5-bisphosphate carboxylase close to the active site

    J. Biol. Chem.

    (2002)
  • W.J. Manning et al.

    Ethylenediurea (EDU): a research tool for assessment and verification of the effects of ground level ozone on plants under natural conditions

    Environ. Pollut.

    (2011)
  • E. Oksanen et al.

    Impacts of increasing ozone on Indian plants

    Environ. Pollut.

    (2013)
  • A.K. Pandey et al.

    Differences in responses of two mustard cultivars to ethylenediurea (EDU) at high ambient ozone concentrations in India

    Agric. Ecosyst. Environ.

    (2014)
  • A.K. Pandey et al.

    Searching for common responsive parameters for ozone tolerance in 18 rice cultivars in India: results from ethylenediurea studies

    Sci. Total Environ.

    (2015)
  • A.K. Pandey et al.

    High variation in resource allocation strategies among 11 Indian wheat (Triticum aestivum) cultivars growing in high ozone environment

    Climate

    (2019)
  • E. Paoletti et al.

    Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): roles of biochemical changes and decreased stomatal conductance in enhancement of growth

    Environ. Pollut.

    (2008)
  • E. Paoletti et al.

    Use of antiozonant ethylenediurea (EDU) in Italy: verification of the effects of ambient ozone on crop plants and trees and investigation of EDU's mode of action

    Environ. Pollut.

    (2009)
  • R. Rai et al.

    Application of ethylene diurea (EDU) in assessing the response of a tropical soybean cultivar to ambient O3: nitrogen metabolism, antioxidants, reproductive development and yield

    Ecotoxicol. Environ. Saf.

    (2015)
  • M. Sharma et al.

    Proteomics unravel the regulating role of salicylic acid in soybean under yield limiting drought stress

    Plant Physiol. Biochem.

    (2018)
  • D. Simpson et al.

    Ozone e the persistent menace: interactions with the N cycle and climate change

    Curr. Opin. Environ. Sustain.

    (2014)
  • S. Singh et al.

    Screening three cultivars of Vigna mungo L. against ozone by application of ethylenediurea (EDU)

    Ecotoxicol. Environ. Saf.

    (2010)
  • S. Singh et al.

    Differential protection of ethylenediurea (EDU) against ambient ozone for five cultivars of tropical wheat

    Environ. Pollut.

    (2009)
  • I.K. Smith et al.

    Assay of glutathione reductase in crude tissue homogenates using 5,50-dithiobis(2-nitrobenzoic acid)

    Anal. Biochem.

    (1988)
  • S. Tiwari et al.

    Protection of palak (Beta vulgaris L. var All green) plants from ozone injury by ethylenediurea (EDU): roles of biochemical and physiological variations in alleviating the adverse impacts

    Chemosphere

    (2009)
  • S. Tiwari et al.

    Effectiveness of different EDU concentrations in ameliorating ozone stress in carrot plants

    Ecotoxicol. Environ. Saf.

    (2010)
  • X.K. Wang et al.

    Assessing the impact of ambient ozone on growth and yield of a rice (Oryza sativa L.) and a wheat (Triticum aestivum L.) cultivar grown in the Yangtze Delta, China, using three rates of application of ethylenediurea (EDU)

    Environ. Pollut.

    (2007)
  • Y.S. Xu et al.

    Mesophyll conductance limitation of photosynthesis in poplar under elevated ozone

    Sci. Total Environ.

    (2019)
  • E. Agathokleous et al.

    Ethylenedi-urea (EDU), an effective phytoproctectant against O3 deleterious effects and a valuable research tool

    J. Agric. Meteorol.

    (2015)
  • G.K. Agrawal et al.

    Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings

    Proteomics

    (2002)
  • N. Ahmad Khan et al.

    Analysis of proteins associated with ozone stress response in soybean cultivars

    Protein Pept. Lett.

    (2013)
  • S.U. Alfonso et al.

    Photosynthetic responses of a C3 and three C4 species of the genus Panicum (s.l.) with different metabolic subtypes to drought stress

    Photosynth. Res.

    (2012)
  • D.I. Arnon

    Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris

    Plant Physiol.

    (1949)
  • M.R. Ashmore

    Assessing the future global impacts of ozone on vegetation

    Plant Cell Environ.

    (2005)
  • J.M. Basahi et al.

    Assessing ambient ozone injury in olive (Olea europaea L.) plants by using the antioxidant ethylenediurea (EDU) in Saudi Arabia

    Environ. Monit. Assess.

    (2016)
  • W.F. Bayer et al.

    Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions

    Anal. Biochem.

    (1987)
  • V. Beyel et al.

    Differential inhibition of photosynthesis during preflowering drought stress in Sorghum bicolor genotypes with different senescence traits

    Physiol. Plantarum

    (2005)
  • A. Bhatia et al.

    Impact of Tropospheric Ozone on Crop Growth and Productivity–A Review

    (2012)
  • S. Bohler et al.

    A DIGE analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism

    Proteomics

    (2007)
  • Cited by (24)

    • Efficiency of protectants in alleviating ozone stress on rice cultivars (Oryza sativa L.)

      2022, Atmospheric Pollution Research
      Citation Excerpt :

      Similarly, while increased nitrogen fertilizer use above the acceptable level protects the plants against O3 damage, a synergistic impact was also observed in pathogen-infected plants (Ghosh et al., 2018). Amongst several methods, application of protectants like ethylene diurea (Singh et al., 2018; Baqasi et al., 2018; Rathore and Chaudhary, 2019; Gupta et al., 2020; Jabeen and Ahmed, 2021; Shang et al., 2022), calcium acetate, calcium chloride (Lakaew et al., 2021a,b), urea (Chaudhary and Rathore, 2020; Gupta and Tiwari, 2020; Shang et al., 2021; Peng et al., 2021), ascorbic acid (Chen and Gallie, 2005; Bellini and De Tullio, 2019), Panchagavya, neem oil (Kovilpillai et al., 2021; JawaharJothi et al., 2022), agrochemicals (Saitanis et al., 2015), arbuscular mycorrhizal fungi (Cui et al., 2013), natural plant extracts (Daripa et al., 2016), sesquiterpenes (Palmer – Young et al., 2015), di – 1 – p – menthene (Agathokleous et al., 2014, 2016; Francini et al., 2011), chlorothalonil (Hassan, 2006), chitosan nanoparticles (Picchi et al., 2021), biochar (Ghosh et al., 2021), have effectively mitigated the O3 stress. Furthermore, pink pigmented facultative methylotrophs (PPFM) are yet another microbial culture that is effective in mitigating various abiotic stress (Sivakumar et al., 2017, 2018; Bajpai et al., 2022).

    • Ethylenediurea offers moderate protection against ozone-induced rice yield loss under high ozone pollution

      2022, Science of the Total Environment
      Citation Excerpt :

      Therefore, it is essential to assess the negative impacts of O3 on food security and provide available information on mitigating the negative impacts. Ethylenediurea (N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea, EDU) is an antiozonant extensively used to evaluate the effects of O3 on vegetation under ambient and elevated O3 conditions (Agathokleous et al., 2021; Chaudhary and Rathore, 2021; Gupta et al., 2020). In addition, EDU is used to screen crop cultivars for O3 tolerance, thus informing cultivation practices at areas with O3 pollution (Pandey et al., 2015; Jiang et al., 2018; Singh et al., 2009; Ashrafuzzaman et al., 2017).

    • Evaluating impacts of biogenic silver nanoparticles and ethylenediurea on wheat (Triticum aestivum L.) against ozone-induced damages

      2022, Environmental Research
      Citation Excerpt :

      Increased chlorophyll contents were observed in AgNPs treated Citrullus lanatus seedlings which promote the higher accumulation of soluble proteins resulting in higher physiological functions (Acharya et al., 2020). The impact of EDU on gas exchange parameters was assessed in plants exposed to ambient O3, and most studies reported that there was no significant effect of EDU on crops (Pandey et al., 2014; Gupta et al., 2018, 2020). Ozone oxidizes several bio-molecules in plant cells by generating ROS, such as peroxidation of lipids that destroy the membrane integrity by altering the lipid structures (Oksanen et al., 2003).

    View all citing articles on Scopus
    View full text