Minimally invasive quantitative tracking of minerals, sugars, amino acids, and hormones in the leaf apoplast of Arabidopsis thaliana and Vicia faba

The apoplast of leaves is involved in nutrient transport, microbe-host interactions, systemic signaling, cell wall dynamics, and serves as an interface for various other physiological processes. The composition of the apoplastic solute pool, which supports many of these functions, is dynamic and shaped by developmental and environmental cues. However, due to the complexity and compartmentalization of the apoplast, analysing these fluids - and thus the associated physiological processes - remains technically challenging. This study introduces a minimally invasive method for extracting apoplastic fluids from leaves of selected dicots (e.g. Arabidopsis thaliana, Vicia faba, and many more), offering two key advantages: (i) repeated extractions from the same leaves to generate time-series data, such as every 24 hours, over consecutive days, and (ii) high spatial resolution, enabling identification of macrodomains within the leaf apoplast. For example, abscisic acid macrodomains were revealed along the leaf axis, providing insight into apoplastic hormone regulation. The method also reveals other previously unrecognized aspects, such as the accumulation of kaempferol glycosides in the apoplast after plants experienced salt stress. Finally, the method addresses the distortion of apoplast compound levels caused by dilution bias that results from the inconsistent recovery of infiltration fluid. Adding pyranine enables correction, ensuring accurate and comparable data. By integrating spatial and temporal precision, this new tool will promote a deeper understanding of plant apoplastic processes and their physiological relevance in various biological contexts.