In vivo monitoring an important plant immune signaling molecule salicylic acid by rhodamine-engineered probes and their density functional theory (DFT) calculations

Abstract

Monitoring the dynamic fluctuations of plant immune signaling molecules is particularly meaningful and challenging in crop protection. Herein, four rhodamine-functionalized probes (F1-F4) were designed and synthesized to attempt to selectively detect a plant hormone salicylic acid (SA). Screening results revealed that probe F1 bearing a 4,5-dimethoxy-2-nitrobenzyl carbamate moiety was extremely sensitive and selective towards SA along with a conspicuous fluorescence “turn-on” manner. The Job’s plot experiment disclosed a 1:1 binding mode together with a binding constant of 1.34 × 104 M−1, indicating that an appreciable hydrogen bonding interaction happened between probe F1 and SA, thereby leading to the spirolactam ring breakage and the succeeding fluorescence generation. Concentration-dependent titration assays offered an available linear relationship for quantifying SA (15–70 μM) and the detection limit of probe F1 to SA was 1 μM. Density functional theory (DFT) calculations displayed that a smaller energy gap (ΔEF1-Ⅱ = 498.89 kJ/mol) was obtained between its lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO), manifesting that probe F1 was more reactive and sensitive than those of probes F2-F4 (ΔE = 567.07 ∼ 601.74 kJ/mol) after adsorption with salicylic acid. Meanwhile, the possible monitoring mechanism was elucidated by 1H NMR titration experiments, probe-SA DFT calculations, and HRMS. Finally, in vivo confocal imaging results found that probe F1 could delicately and selectively monitor SA on the roots of cucumber. This study can motivate the intensive exploration of multitudinous fluorescent probes for direct SA monitoring in vivo.

Keywords

• Rhodamine probes
• Carbamate moiety
• Salicylic acid
• DFT calculations
• In vivo imaging