ROS-scavenging Mn₃O₄ nanozymes increase cotton alkaline tolerance by increasing the stability of actin filaments and reducing Na⁺ content

Alkaline stress in saline soil limits cotton production that may be improved by using emerging nanobiotechnology approaches. Here, we applied poly acrylic acid coated Mn₃O₄ nanoparticles (PMO) on cotton leaves which showed higher chlorophyll content (up to 100.0%) and fresh weight (46.9%) and lower electrolyte leakage rate (up to 6.8%) and cell death rate (up to 84.8%) than controls. Further investigation showed that PMO can maintain reactive oxygen species (ROS) homeostasis, increase the stability of actin filament (AF), and reduce Na⁺ content. Confocal imaging and ROS content measurement showed that PMO foliar application effectively alleviated ROS over-accumulation (up to 16.4% decrease for H₂O₂ and 45.3% decrease for O₂^•-) in cotton leaves. Moreover, under alkaline stress, genes for AF depolymerization such as GhADF1/8 and GhADF6 and for AF polymerization such as GhADF5 were significantly down-regulated in PMO treated cotton lines relative to those in the control, consistent with the fluorescence intensities of AFs. Furthermore, our results showed that PMO mitigated Na⁺ toxicity under alkaline stress, as indicated by the reduced Na⁺ fluorescence intensity and Na⁺ content. Furthermore, relative to those of the control, PMO treatment increased seed yield and lint yield by 65.0% and 66.3% respectively. Together, our work demonstrates that ROS scavenging PMO alleviated alkaline stress by stabilizing actin filaments and reducing Na⁺ toxicity.