Use of <scp>Mn₃O₄</scp> nanozyme to improve cotton salt tolerance

Abstract

Cotton is an important fibre and oil crop across the globe. Salinity is a global issue limiting efficient crop production. Especially, salinity is a big threat for cotton production in China, since the majority of cotton are planted in Xinjiang province which has soil salinization issue. Nowadays, more than 80% cotton in China was harvested from Xinjiang province which is a semi-arid area with severe salinity issue (Feng et al., 2017). Many approaches were tried to improve cotton production in saline land, including proper irrigation, breeding salt-tolerant cotton species and flushing saline soil with fresh water (Sara Francisco Costa et al., 2018). However, all these approaches have its own limitations. For example, breeding programmes take long time and flushing saline soil with fresh water is not affordable in semi-arid area. New approach such as nanobiotechnology could give alternative solutions to improve plant salt tolerance. To facilitate the adoption of nanobiotechnology in agriculture, learning from nature are encouraged. For example, ROS over-accumulation is a secondary stress in crop plants under salinity and results in oxidative damage on macro-molecules such as DNA, protein and lipids in plants (Mittler, 2017). Increasing ROS scavenging ability has been proved as an effective way to improve plant salt tolerance (Wu et al., 2017). Thus, the use of nanomaterials with good ROS scavenging ability might help to improve plant salt tolerance. Indeed, nano-improved plant salt tolerance has been reported in many plant species, including rice, rapeseed, Arabidopsis and cotton (Wu and Li, 2022). While, to date, the nanoparticles used to improve cotton salt tolerance are nanoceria (An et al., 2020) and nano-zinc (Hussein and Abou-Baker, 2018). The former one has catalytic ROS scavenging ability while the safety concern about cerium is always an issue. The latter one is formed with essential micronutrients for plants but is lacking of catalytic ROS scavenging ability. Thus, designing nanomaterials that have the advantages of being essential nutrients for plants and good ROS scavenging ability could be a good strategy to improve plant salt tolerance. Manganese (Mn) is an essential micronutrient for plants and plays important role in plant growth. Mn is known as a critical component of the photosynthetic system and directly participates in the hydrolysis process (Barber, 2017). Also, Mn is important for the activities of various enzymes (Emsley, 2013), for example antioxidant enzyme Mn-SOD (superoxide dismutase) requires Mn as cofactor. Thus, not surprisingly, Mn fertilizers are widely used in agriculture. Thus, Mn-based nanomaterials with good ROS scavenging ability could be a good candidate to improve cotton salt tolerance. In this work, we designed and synthesized a PAA (polyacrylic acid) coated Mn3O4 nanoparticles (PAA@Mn3O4 nanoparticles, PMO). HR-TEM (high resolution-transmission electron microscopy) images showed that PMO are well dispersed and the TEM size of PMO is 5.6 ± 1.1 nm (Figure 1a). The hydrodynamic diameter and zeta potential of PMO are 9.2 ± 1.2 nm and − 38.3 ± 0.1 mV (Figure 1b). The size and charge are good to ensure its delivery efficiency in plants (Wong et al., 2016) (Wu et al., 2017). XRD (X-ray diffraction) analysis further confirmed that PMO are Mn3O4 nanoparticles (Figure 1c). XPS (X-ray photoelectron spectroscopy) results shows that the ratio of Mn2+/Mn3+/Mn4+ in PMO is 0.4:1:1.9 (Figure 1d). In vitro ROS scavenging test shows that the ability to scavenge OH•, O2• —, and H2O2 is 95.1%, 30.0%, and 6.7% for PMO (Figure 1e). Similar to our previous work (Liu et al., 2021), PMO was foliar delivered (with 0.05% Silwet L-77) to cotton plants (two true leaf stage). Confocal imaging results showed that compared with no nanoparticle control, under salt stress, PMO-treated cotton plants showed significant lower fluorescent intensity of DCF (2′,7′-dichlorofluorescein) dye (indicating H2O2) (1.9 ± 0.2 vs 12.7 ± 3.4), DHE (dihydroethidium) dye (indicating O2• —) (1.8 ± 0.1 vs 1.3 ± 0.1) and HPF (hydroxyphenyl fluorescein) dye (indicating OH•) (3.8 ± 0.9 vs 9.2 ± 1.1) (Figure 1f,g). These results further confirmed that PMO has good ROS scavenging ability to help to maintain ROS homeostasis in cotton plants. It suggests that by addressing the nature of essential micronutrients and good ROS scavenging ability, PMO can be a good candidate to improve cotton salt tolerance. To investigate the role of PMO in improving cotton salt tolerance, we treated cotton plants (second true leaf stage) with foliar delivered 200 mg/L PMO and then subjected it to salinity (200 mM NaCl) for 5 days. Cotton plants treated with MnCl2 (200 mg/L) and 0.05% Silwet L-77 were used as positive control and negative control, respectively. After 5 days' salt stress, compared with control plants, obvious improvement of phenotypic performance was observed in cotton plants treated with PMO (Figure 1h). This is in accordance with fresh weight data. Under salt stress, PMO-treated cotton plants showed significant higher fresh weight (2.9 ± 0.1 g) than control plants (1.7 ± 0.1 g) (Figure 1i). These results confirmed that PAA@Mn3O4 nanoparticles with proper ROS scavenging ability are good candidate to improve cotton salt tolerance. Moreover, knowing the molecular mechanisms underlining nano-improved plant salt tolerance is of importance to give clue to further studies. K+/Na+ ratio is a hallmark for plant salt tolerance. Here, using APG2 (K+ fluorescent dye) and CoroNa Green (Na+ fluorescent dye), we investigated subcellular distribution of K+ and Na+ in leaf mesophyll cells of salt-stressed control plants and cotton plants treated with PMO via confocal microscopy imaging. Confocal imaging results showed that compared with control plants, PMO-treated cotton plants maintained more K+ in mesophyll cells under salt stress (Figure 1j,k). This is confirmed by the calculated K+ fluorescent dye intensity, showing that under salinity stress, PMO has significantly higher cytosolic (43.5 ± 4.3) and vacuolar (38.3 ± 4.5) K+ fluorescent intensity than control plants (8.5 ± 0.7 for the cytosol and 14.7 ± 1.7 for the vacuole) (Figure 1l). Similarly, compared with control plants (35.4 ± 1.7 and 26.6 ± 4.1 for cytosolic and vacuolar Na+), PMO-treated cotton plants showed significantly lower Na+ fluorescent intensity in the cytosol (12.1 ± 0.2) and the vacuole (13.6 ± 2.6) (Figure 1l). The calculated associated cytosolic K+/Na+ ratio for control and PMO-treated cotton plants under salt stress is 0.24 and 3.60, respectively (Figure 1l). In this study, we showed that with proper control of size, charge, composition and ROS scavenging ability, the designed PMO (PAA@Mn3O4 nanoparticles) improved salt tolerance in cotton. Also, our previous work showed that the used PMO does not have negative effect on rat heart, liver, spleen, lung and kidney (Shan et al., 2023), suggesting PMO is an environmental friendly and bio-safe nanomaterial. Whether PMO are good candidate to improve abiotic stress tolerance in broad plant species is worthy to be further studied. Our previous work showed that cerium oxide nanoparticles do not enter into the seeds of rice plants (Zhou et al., 2021). Whether PMO will accumulate in seeds or other vegetative parts of plants still needs to be investigated. Moreover, our results showed that Mn3O4 nanoparticles can help to maintain ROS homeostasis and K+/Na+ ratio in cotton plants under salt stress. These newly explored mechanisms for Mn3O4 nanoparticles could give clue to better design Mn-based nanomaterials for nano-enabled agriculture. Overall, our work suggests that learning from nature and designing nanomaterials with desired properties are important factors to facilitate the adoption of nanobiotechnology in agricultural production. Future studies are encouraged to explore more nanomaterials with controlled properties for agricultural applications. This work was supported by the National Key Research and Development Program of China (2022YFD2300205), the NSFC grant (No. 32071971, 31901464) and the Hubei Agricultural Science and Technology Innovation Center Program (2021-620-000-001-032) to H.W. The authors declare no competing financial interests.

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