Research articleGraphene ameliorates saline-alkaline stress-induced damage and improves growth and tolerance in alfalfa (Medicago sativa L.)
Introduction
Soil salinization and alkalization is a major global environmental concern that not only limits plant growth and development, but also contributes to land degradation. For instance, the world's arable land area is 1.5 × 109 ha, but 23% (0.34 × 109 ha) of the area is saline, and 37% (0.56 × 109 ha) is sodic. Neutral salts (NaCl and Na2SO4) cause salt stress, whereas alkali stress is caused by alkaline salts (NaHCO3 and Na2CO3) (Yang et al., 2007). Alkaline salt stress is best defined as alkali stress in general, whereas salt stress refers to neutral salt stress only. To survive under those adverse conditions, via a series of dramatic physiological and biochemical changes, plants can use complex defensive mechanisms (Wu et al., 2018). Soil salinity and alkalinity initially restrict plant growth in the form of osmotic stress and later induce ionic stress (Munns and Tester, 2008). During the initial step, cell expansion, cell wall synthesis, protein synthesis and stomatal conductance are all inhibited (Apel and Hirt, 2004). The later step of ion aggregation promotes the generation of reactive oxygen species (ROS). In mitigating oxidative damage, antioxidant enzymes, including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and ascorbate peroxidase (APX), are important components of the ROS scavenging method (Wang et al., 2016). Moreover, photosynthetic activity in plant leaves also will be reduced under salt or alkali stress. For instance, salt stress can damage light-capturing pigment complexes on photosystem I and photosystem II (LHC I and LHC II), resulting in the reduced chloroplast's ability to absorb light and transfer electrons, and reduce energy conversion effects, which ultimately results in the reduced photosynthesis' absorption, transmission and conversion of energy efficiency (Mu and Chen, 2020).
The use of nano technology, which can be effective and economical in contrast with traditional methods, is a new age in the field of plant fertilization and environmental remediation (Sabet and Mortazaeinezhad, 2018). Studies have shown that the nano-scale use of nutrients (nano-fertilization) can improve the efficiency of nutrient use and lead to less fertilizer use, increased yield production, and decreased environmental pollution (Chen and Yada, 2011, Husen and Siddiqi, 2014, Khot et al., 2012). A growing number of studies have shown that nanoparticles have positive effects on plants, such as promoting seed germination and plant development (Chen and Yada, 2011), improving nitrogen metabolism and plant photosynthesis (Husen and Siddiqi, 2014), promoting nutrient absorption, and fertilizer utilization rates (Khot et al., 2012). Graphene, a popular two-dimensional carbon-based nanomaterial, has received increasing attention due to its unique physical and structural properties (Avouris, 2010). Low sulfonated graphene concentration may scavenge ROS in the roots, change root morphology and improve the health of maize seedlings (Ren et al., 2016). High graphene concentrations inhibit rice growth, eventually reducing its biomass (Liu et al., 2015). Some studies also summarized the potential key mechanisms of phytotoxicity of graphene family nanomaterials, include physical effects (mechanical damage and physical obstruction) and biochemical and physiological effects (increase in performance of reactive oxygen species (ROS), antioxidant activity generation and inhibition, metabolic disorders, and photosynthesis suppression by decreasing chlorophyll biosynthesis) (Wang et al., 2019). However the influence mechanism of nanomaterials on plants is complicated, as it is not only related to class, concentration and surface characteristics of nanomaterials, but also to types of plants and seeds size (Wu et al., 2012), there are limited studies on the specific application rate in the saline-alkaline soil which are because of different mobility and accessibility to plants.
Alfalfa (Medicago sativa L.) is widely distributed worldwide with high protein, vitamin, and mineral contents and is predominantly located in the northern, northwestern, and northeastern regions of China (Jiang et al., 2019, Peng et al., 2008). However, in those regions, soil salinization is significantly increasing, reducing the productivity and persistence of alfalfa (Munns and Tester, 2008, Yang et al., 2007). Therefore, for the use of these saline areas, breeding and finding salt-alkali tolerant alfalfa varieties is important. Using the relatively salt-tolerant alfalfa Zhongmu NO.1, we analyzed the responses of Zhongmu NO.1 to salt and alkali stresses under graphene (Nano-C) treatment at both in terms of physiological and morphological responses. And, we used the two main factors, Nano-C and pH, to construct the models. The main objectives were (1) to evaluate the effects of non-uniform Nano-C treatments on the growth of alfalfa; (2) to evaluate the effects of Nano-C on the morphological and physiological regulation of the leaves and roots under salt and alkali stresses, and (3) to investigate the mechanisms of coupling effects and develop optimal Nano-C under salt-alkaline stress by constructing suitable models.
Section snippets
Plant material preparation
Alfalfa seeds (Zhongmu NO.1) were purchased from Barenbrug (Tianjin, China) Co., Ltd. The seeds were surface-sterilized with 75% ethyl alcohol for 30 s and 5% sodium hypochlorite solution for 15 min, then thoroughly washed 5 times. The seeds were pregerminated in a light growth chamber (light 24 /dark 18 °C, 16 /8 h) on wet filter paper and transplanted at the point they reached the cotyledon stage with 4–5 cm long taproots. The matrix, using a 3:1:1 mixture of peat, vermiculite and perlite,
Significant effects of the Nano-C
Before the stress treatments, with increasing Nano-C level (from 0 to 20 g kg−1) in the mixed matrix, DW and FW of alfalfa showed that a downward trend after an initial increase, and the analysis of variance of FW showed that the effects of 5 g kg−1 Nano-C treatment were significantly increased compared with CK treatment (p < 0.05, Fig. 1B). Dry weight was also increased at the lower Nano-C level (5 g kg−1) (p > 0.05, Fig. 1A). After the stress treatments, DW and FW of S-CK and A-CK treatments
Discussion
Excess saline-alkali stress results in osmotic stress and ion toxicity, which inhibits the growth of alfalfa, reduces biomass, increases the content of Na+ plant tissue, decreases K+ content, increases peroxidation of the cell membrane and inhibits photosynthesis, especially under high pH conditions (Apel and Hirt, 2004, Wu et al., 2018). The pH benefit is mediated by the extreme effects of salinity and alkalinity on plant growth, which can directly affect plant roots, alter nutrient
Conclusion
Our findings suggest the oxygen free radicals generated by intermediate Nano-C concentrations (5g kg-1) can be used as signal molecules to induce gene expression in the antioxidant enzyme systems through cell signal transduction and promote SOD, CAT and POD biosynthesis in alfalfa roots and leaves, relieving damage caused by saline-alkali stress. Higher Nano-C concentration (10 and 20 g kg−1) will damage the protective enzyme system and cell structure. Our isozyme outcomes showed how
Author contributions
ZC designed and performed the experiments, and wrote manuscript. QW analyzed the data and revised manuscript. All authors have read and approved the final version of the manuscript.
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.
Acknowledgment
This study was supported by National Key R&D Program of China (2017YFE0111000).
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