A near-infrared ratiometric fluorescent probe for visual sensing of H2S and monitoring its fluctuation in plant roots under drought and flooding stresses.

BACKGROUND: Hydrogen sulfide (H2S) is a key endogenous gasotransmitter involved in plant physiological regulation and stress responses. Monitoring its dynamic changes in plants is essential for understanding related signaling mechanisms. However, due to its chemical instability and the complexity of plant tissues, developing a reliable method for accurate quantification and real-time visualization of H2S in living plants remains challenging. Although fluorescent probes have been developed for H2S imaging, most still face critical limitations, such as emission in the visible region being susceptible to background fluorescence interference, and insufficient quantitative reliability of single-wavelength-based signal output modes in complex samples. Therefore, developing novel probes with near-infrared emission and rationetric response characteristics is of great significance.

RESULTS: We constructed a near-infrared rationetric fluorescent probe, NIR-Cou-H2S, for H2S detection. The probe itself emits at 716 nm, and after specific reaction with H2S, a new emission peak appears at 552 nm, resulting in a distinct dual-emission rationetric response (716 nm/552 nm) accompanied by a visible color change. Using chemometrics-based fluorescence analysis, the probe successfully enabled direct quantitative detection of H2S in river and lake water samples. Its near-infrared emission effectively reduced interference from plant autofluorescence, thereby achieving high-contrast dual-channel fluorescence imaging. The probe was successfully applied for high-quality in situ visualization of H2S in living cells and tobacco seedling roots. More importantly, using NIR-Cou-H2S, we observed and recorded in real time the dynamic upregulation trend of endogenous H2S levels in tobacco roots under both drought and flooding stress conditions.

SIGNIFICANCE: As a novel near-infrared rationetric probe, NIR-Cou-H2S provides a powerful tool for monitoring endogenous H2S dynamics in plants. Its characteristics significantly improve the reliability of imaging and quantification in complex plant samples, offering a key methodological approach for further elucidating the regulatory mechanisms of H2S in plant stress resistance.