Quantitative tools for analyzing rhizosphere pH dynamics: localized and integrated approaches.

The rhizosphere, the region surrounding plant roots, plays a critical role in nutrient acquisition, root development, and plant-soil interactions. Spatial variations in rhizosphere pH along the root axis are shaped by environmental cues, nutrient availability, microbial activity, and root growth patterns. Precise detection and quantification of these pH changes are essential for understanding plant plasticity and nutrient efficiency. Here, we present a refined methodology integrating pH indicator bromocresol purple with a rapid, non-destructive electrode-based system to visualize and quantify pH variations along the root axis, enabling high-resolution and scalable monitoring of root-induced pH changes in the rhizosphere. Using this approach, we investigated the impact of iron (Fe) availability on rhizosphere pH dynamics in wild-type (WT) and bHLH39-overexpressing (39Ox) seedlings. bHLH39, a key basic helix-loop-helix transcription factor in Fe uptake, enhances Fe acquisition when overexpressed, often leading to Fe toxicity and reduced root growth under Fe-sufficient conditions. However, its role in root-mediated acidification remains unclear. Our findings reveal that 39Ox plants exhibit enhanced rhizosphere acidification, whereas WT roots display zone-specific pH responses depending on Fe availability. To refine pH measurements, we developed two complementary electrode-based methodologies: localized rhizosphere pH change for region-specific assessment and integrated rhizosphere pH change for net root system variation. These techniques improve resolution, accuracy, and efficiency in large-scale experiments, providing robust tools for investigating natural and genetic variations in rhizosphere pH regulation and their role in nutrient mobilization and ecological adaptation.