Research PaperNanomaterial sulfonated graphene oxide advances the tolerance against nitrate and ammonium toxicity by regulating chloroplastic redox balance, photochemistry of photosystems and antioxidant capacity in Triticum aestivum
Graphical Abstract
Introduction
Graphene or graphene oxide (GO), classified as a two-dimensional carbon nanomaterial, has interdisciplinary usage in different fields such as biosensors, biomedical applications, tissue engineering, drug delivery, pesticide application, and energy storage (Verma et al., 2019). Especially in plant-based studies, GO releases to irrigation water and soil during application processes (Zhao et al., 2014). For environmental protection, the ecological and health risks of GO exposure should be evaluated. The phytotoxicity of GO is expressed on the loss of growth index, germination rate, water content of seeds and increases the ratio of unsaturated to saturated fatty acids in plants (Hu et al., 2014). GO causes damages in photosynthetic systems of wheat seedlings subjected to cadmium stress, as proved by inhibition of PSII efficiency, photosynthetic rate and stomatal conductance (Gao et al., 2019). The same effect is reported by Chen et al. (2019) that the pea plants subjected to the reduced graphene exhibit the inhibited activity of photosystem II (PSII) under control conditions. These GO responses are related to exposure concentrations/times, development stages and growing media of plants, including hydroponics and soil culture (Wang et al., 2019). Conversely, the positive responses of GO reported that graphene is effective for the suppression of inactivated aquaporin genes under salt stress (Pandey et al., 2018). After GO exposure to Vicia faba (400 and 800 mg L−1), reactive oxygen species (ROS) production and the oxidation of lipids and proteins decreased (Anjum et al., 2014). Zhang et al. (2015) showed that graphene improves the germination and growth of tomatoes. Graphene nanosheets remove oxidative status by regulating total antioxidant enzymes and secondary metabolites in Capsicum annuum and Solanum melongena (Younes et al., 2019). These contradictory findings represent that upward research on the connection with GO, defense system, and metabolic process is needed in plants with/without stress.
GO is functionalized by grafting ligands or binding of sulfonic acid, epoxy, thiol, hydroxyl, and carboxyl functional groups or coating with polyethylene glycol in pre- and post-synthesis steps of processing. Therefore, after this process, the changes in the shape, surface, and size of GO advance its applicability and adsorption of capacity and, decrease toxicity (Kanth et al., 2020). According to the difference of the side groups, graphene diversifies as pristine graphene, graphene oxide, sulfonated graphene, and fluorographene (Ren et al., 2016). There are different responses between functionalized and non-functionalized forms of GO in plants (Cañas et al., 2008). For example, epoxy or positively charged amine groups in GO contribute to exciton formation, which plays an important role in photosynthesis (Sharma et al., 2020). In addition, GO is removed excess levels of the contaminants such as cadmium in an aqueous solution by inducing adsorption capacity (Tan et al., 2016). Sulfonated graphene oxide (SGO), modified with the sulfonic group, has high dispersibility levels in an aqueous solution, and adsorption capability and is more physiologically stable (Ren et al., 2018, Thombal and Jadhav, 2016). Among nanomaterials, SGO is a biocompatible candidate for tolerance against stress conditions due to these properties. However, no information is available on the direct interactions of SGO on tolerance responses in stress-treated plants.
The sustainability of photosynthesis is a critical phenomenon for plant survival under stress conditions. Stress, including salt, drought, cold, and nutrient deficiency/excess causes a limitation of growth, energy metabolism in different cellular compartments (García-Caparrós et al., 2020), especially on chloroplasts. Nitrogen (N2), one of the essential macronutrients, plays a significant role in metabolic reactions associated with the growth and development of plants. Nitrate (NO3−) and ammonium (NH4+) are the absorption and utilization forms of inorganic N2. When NO3−- and NH4+-based N fertilizers are intensively applied to crops for increase quality and production, they produce environmental problems (eutrophication of water reservoirs, contamination and atmospheric pollution) (Esteban et al., 2016). NO3− toxicity results in the leaf chlorosis, reduced CO2 assimilation, photorespiration stimulation, photosynthetic activity disruption triggered by reactive oxygen species (ROS) accumulation and imbalance of carbohydrate metabolism (Bittsánszky et al., 2015). Stress-induced ROS accumulation is regulated via antioxidant mechanisms such as superoxide dismutase (SOD), peroxidase (POX), the ascorbate-glutathione cycle (AsA-GSH), including ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), ascorbate (AsA) and glutathione (GSH) (Ghorbanpour et al., 2013). Also, to remove these negative effects on chloroplasts, nanomaterials generally have a role in activating of defense system by enhancing carboxylation of Rubisco and light absorption capacity of chloroplasts (Ze et al., 2011), electron transport rate, and inhibition of ROS generation in the chloroplast (Giraldo et al., 2014). After exposure to 100 μg L−1 graphene, the chloroplasts of rice plants contain about 44% of the accumulated graphene located in the thylakoids (Lu et al., 2020). The same researchers detected that when graphene was present, ATP production increased in the chloroplasts of rice plants by 1.38-fold compared to untreated chloroplasts. Lu et al. (2020) hypothesized that the degradation rate of PSII is suppressed under light irradiation by exogenously applied graphene. On the other hand, the modulations of SGO on organellar regulation have yet to be clarified under stress conditions. Although GO and its derivatives can induce the total activity of the antioxidant system against stress in plants, the SGO-mediated scavenging capacity and possible improvement in the photochemistry of photosystems involved in chloroplasts require further elucidation under NS or AS stresses. We hypothesize that SGO can give very different defense responses from those under GO applications in the isolated chloroplasts because SGO contains a functional group with sulfonate. To test this hypothesis, the current study focuses on the effect of SGO on the photochemical reactions and phenomenological fluxes in photosynthetic machinery and the transcript levels of genes encoding photosystems-associated proteins such as psaA (photosystem I P700 chlorophyll a apoprotein A1), psaB (photosystem I P700 chlorophyll a apoprotein A2), psbA (photosystem II protein D1) and psbD (photosystem II protein D2) as well as chloroplastic antioxidant enzyme/non-enzyme activity in Triticum aestivum.
Section snippets
Plant material and experimental design
After the germination, wheat seeds (Triticum aestivum L. cv. Ekiz) were transferred to Hoagland solution under controlled conditions (16/8 h light/dark regime at 24 °C, 70% relative humidity, and 350 μmol m−2s−1 photosynthetic photon flux density) for 21 d. Hoagland solution according to Xu et al. (2008) containing 2.5 mM Ca(NO3)2, 5 mM KNO3, 0.78 mM KH2PO4, 2 mM MgSO4, 29.6 μM H3BO3, 10 μM MnSO4, 50 μM Fe-EDTA, 1.0 μM ZnSO4, 0.05 μM H2MoO4, 0.95 μM CuSO4. For determination of toxicity levels
HR-TEM and EDS analysis of prepared materials
HR-TEM and EDS elemental mapping analysis of prepared pure GO and pure SGO were performed for analyzing the morphology and impact of sulfonyl functionality groups on the obtained SGO. As seen in Fig. 1A, pure GO showed a nanosheets-like structure in the HR-TEM image. The HR-TEM image of SGO showed that the sheets were randomly oriented and crumpled sheets (Fig. 1C). Additionally, compositional analysis by energy-dispersive X-ray spectroscopy (EDS)-elemental mapping, represented in Fig. 1B, 1D.
SEM-EDX analysis of prepared materials
Discussion
The HR-TEM observation presented that the GO fragments were heterogeneous with sharp, irregular edges (Fig. 1A). HR-TEM images obtained in Fig. 1C disclosed that successful modification of GO into SGO was occurred without damaging the spinal nanostructure of graphene oxide. Modified SGO exhibited the sulfonation of GO led to further exfoliation of the clusters of sheets into a random pile. Fig. 1B indicated the presence of C and O elements in the core (red) and green while in Fig. 1D, S
Conclusion
SGOs improved the structural stability, efficiency and photochemical reaction of PSI and PSII impaired by the excess NO3− and/or NH4+ treatments in the wheat chloroplasts. Stress hampered the expression levels of reaction center proteins related to photosystems (psaA, psaB, psbA, and psbD). However, SGO induced the transcript levels of these genes promoting the turnover and repairing of damaged PSI-PSII proteins. All SGO concentrations exhibited the scavenging radical triggered by NS stress by
CRediT authorship contribution statement
C.O.K., E.Y., M.K. and I.T. designed experiments; E.Y. and B.A, F.N.A and F.A. carried out data analysis; H.T. synthesized GO and SGO and analyzed the SGO characterization. C.O.K. and E.Y. interpreted the results and wrote up the first draft of the manuscript; C.O.K. and E.Y. and I.T. critically edited the whole manuscript. All authors read and approved the final manuscript.
Author contributions
Evren Yildiztugay, Ceyda Ozfidan-Konakci, Ismail Turkan: Conceptualization, Methodology, Software Evren Yildiztugay, Ceyda Ozfidan-Konakci: Data curation, Writing – original draft preparation. Busra Arikan, Fatma Nur Alp, Fevzi Elbasan: Investigation, Validation. Ismail Turkan, Mustafa Kucukoduk: Project administration. Ismail Turkan: Supervision. Evren Yildiztugay, Ceyda Ozfidan-Konakci, Halit Cavusoglu, Ismail Turkan: Writing – review & editing.
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.
Acknowledgements
The authors would like to acknowledge to Advanced Technology Research and Application Center of Selcuk University for their kind help regarding the analysis study (XRD). This work was supported by Selcuk University Scientific Research Projects Coordinating Office (Grant Number: 20401149).
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