Monitoring Fluctuations Of H₂O₂ In Plant Under Abiotic Stresses With AIE Probe

As one important reactive oxygen specie(ROS),hydrogen peroxide(H2O2)serves as a key signal molecule related to plant stress response and health status.Therefore,it is of great significance to accurately detect the fluctuation of H2O2 content in plant for monitoring plant health.Various strategies,including chemiluminescent,spectrophotometry and electrochemical methods,have been developed for H2O2 detection.Although these methods can accurately detect H2O2 content,several drawbacks,such as poor reversibility,inability of in-situ real-time detection,limit their practical application.Moreover,professional technical operation and specialized equipment are usually required in these methods.In this paper,based on the characteristics of aggregation-induced fluorescence(AIE)molecules,five groups of fluorescent probes were designed and synthesized by merging H2O2-sensitive trialkylamine moiety to the core fluorophore tetraphenylvinyl(TPE)framework.And a fluorescent chemical probe,named as 2E2F,was selected to detect the fluctuation of H2O2 in plants.In this study,the H2O2 fluorescence probe was applied to severial plant materials,including Malus hupenensis seedlings,‘Orin'apple calli,tobacco(Nicotiana benthamiana),to explore their capacity of tracking H2O2 content fluctuation under different abiotic stresses(mechanical injury,salt stress,strong light stress and drought stresses).The main research contents are as follows:1.Five novel fluorescent molecular probes for H2O2 detection and their corresponding nitrogen oxides(N-oxide)were designed by merging H2O2-sensitive trialkylamine moieties to tetraphenylethene skeleton.Among these probes,2E2F molecule present excellent sensitivity for H2O2 dection.For 2E2F solution,a strong green fluorescence was observed under 365 nm UV light irradiation.And the green fluorescence emission is quenched up to 52%by 100?M H2O2 by being oxidized to a non-fluorescent 2E2F N-oxide(nitrogen oxide).Furthermore,within the range of H₂O₂ content in plant cells(10-100?M),the fluorescence intensity of 2E2F molecule showed a negative linear relationship with H2O2 concentration.2.2E2F has a specific response to H2O2.No fluorescence response was observed when2E2F was exposed to other ROS and their scavengers,common free radical active substances and other metabolites(hormones,amino acids,antioxidants,etc.).Therefore,tracking H2O2wave by 2E2F can't be interfered by other substances in plant.3.2E2F presents good reversibly for H₂O₂ dection.After incubating the‘Orin'apple calli or Malus hupenensis seedlings with 2E2F compound or the corresponding 2E2F N-oxide,strong green fluorescence was observed under UV light,and quenched by salt stress treatment-induced H2O2 enhancement.When salt stress was cleared,the fluorescence was recovered.Therefore,the fluctuation of H2O2 can be detected throught the mutual transformation between2E2F with 2E2F N-oxide.4.2E2F can detect the fluctuation of H2O2 under different abiotic stresses.In response to various abiotic stresses,including mechanical damage,high salt,strong light and drought,fluorescence fluctuations,responding to H₂O₂ concentration alterations in vivo,were visible to the naked eye under irradiation of commercial available UV light(365 nm)after simple injection of this H2O2 probe solution into seedling leaves.Moreover,the green fluorescence recovered after rehydration on drought-treated tobacco.Notably,this method is ease of operation,and no skillful operation and professional equipment were required.In summary,five groups of compounds with AIE properties were designed in this study,and 2E2F molecules were selected as probes to monitor the fluctuation of H2O2 in plants.It is a sensitive,selective and reversible green fluorescent chemical probe that can in situ real-time monitor the fluctuations of H2O2 in plants to assess the health status of plants without the aid of skillful operation and specialized equipment.This study provides a novel fluorescence imaging tool for dynamic tracking of H2O2 in vivo,with good potential as an early sensor of plant stress health.