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

The leaf apoplast is involved in nutrient transport, microbe-host interactions, systemic signaling, and cell wall dynamics, serving 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, analyzing these fluids-and thus the associated physiological processes-remains technically challenging. We introduce 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 h, 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 aspects, such as the accumulation of kaempferol glycosides in the apoplast after salt stress. Finally, the method addresses the distortion of apoplast compound levels caused by dilution bias resulting 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.