Green fluorescent FM dyes with prolonged retention for 4D tracking of plasma membrane dynamics in live plants under...

Macroscopic phenotypic changes in plants are frequently employed as a means of evaluating the biological response of plants to external environmental stresses. However, the lack of effective observational tools at the microscopic cellular level hinders the ability to fully comprehend the intricacies of this response. Herein, we developed a plasma membrane fluorescent dye with target-activated green emission complemented with conventional FM dyes, and established a four-dimensional (4D) imaging approach based on this dye for spatio-temporal monitoring of plasma membrane dynamics during cellular responses to external environmental stress. A green fluorescent dye, designated FMG-DBO, was constructed by modifying the bridged unit between the aniline donor and the pyridinium acceptor. Its green emission can be combined with that of conventional FM dyes, enabling high-resolution imaging of plant leaf cells containing chlorophyll. The anchoring ability of the dyes was enhanced by incorporating a rigid diaza[2.2.2]octane unit as an anti-permeability group. The long retention time of the FMG-DBO dye in the plasma membrane enables the tracking of three-dimensional dynamics of the plasma membrane of plant cells. Consequently, an FMG-DBO-based four-dimensional imaging approach was established to monitor dynamic changes of plant cells under external environmental stress at the cellular level. The biological responses of two different drought-tolerant rice root cells to drought stress were examined by this four-dimensional imaging approach. It was observed that the two types of rice root cells exhibited disparate responses to the drought environmen. This approach offers alternative cell-level visualization tools for evaluating the biological responses of plant cells under environmental stress.