Observing root growth and signalling responses to stress gradients and pathogens using the bi-directional dual-flow RootChip

Plants respond to environmental stressors with adaptive changes in growth and development. Central to these responses is the role of calcium (Ca²⁺) as a key secondary messenger. Here, the bi-directional dual-flow RootChip (bi-dfRC) microfluidic platform was used to study defence signalling and root growth. By introducing salinity as sodium chloride (NaCl) treatment via a multiplexed media delivery system (MMDS), dynamic gradients were created, mimicking natural environmental fluctuations. Signal analysis in Arabidopsis thaliana plants showed that the Ca²⁺ burst indicated by the G-CaMP3 was concentration dependent. A Ca²⁺ burst initiated in response to salinity increase, specifically within the stele tissue, for 30 seconds. The signal then intensified in epidermal cells directly in contact with the stressor, spreading directionally towards the root tip, over 5 minutes. Inhibition of propidium iodide (PI) stain transport through the xylem was observed following salinity increase, contrasting with flow observed under control conditions. The interaction of Phytophthora capsici zoospores with A. thaliana roots was also studied. An immediate directional Ca²⁺ signal was observed during early pathogen recognition, while a gradual, non-directional increase was observed in Orp1_roGFP fluorescent H₂O₂ levels, over 30 min. By adjusting the dimensions of the bi-dfRC, plants with varying root architectures were subjected to growth analysis. Growth reduction was observed in A. thaliana and Nicotiana benthamiana roots when exposed to salinity induced by 100 mM NaCl, while Solanum lycopersicum exhibited growth increase over 90 minutes at the same NaCl concentration. Furthermore, novel insights into force sensing in roots were gained through the engineering of displaceable pillars into the bi-dfRC channel. These findings highlight the vital role of controlling fluid flow in microfluidic channels in advancing our understanding of root physiology under stress conditions.