Radial Water Loss Assay in Rice Roots Grown in Different Media

Plants growing in soil are exposed to several environmental stresses, such as soil compaction, flooding, or drought, and need to quickly acclimate to these conditions to survive. Such acclimations include suberization and lignification of the outer and inner parts of the root. Visualization of suberin and lignin depositions in roots can be done with several well-established staining techniques followed by microscopy, but these methods are mostly qualitative. Radial water loss assays provide a quick, robust, and quantitative method to evaluate the formation of suberized and/or lignified outer apoplastic barriers and how tightly they regulate water loss. Here, we provide a detailed step-by-step protocol, from harvesting the roots from soil conditions and cleaning them, to preparing them for water loss measurements, while using basic lab equipment. Since the data obtained is water loss as mass, quantitative and statistical comparisons can be performed. Finally, while in this protocol roots were grown in soil, the method can be applied to roots growing in hydroponics, agar plates, or any other growth conditions that allow non-destructive harvesting of roots. In summary, the protocol provides a fast and reliable method to assess differences in outer apoplastic barrier development of root tissues, allowing for fast root tissue screenings while obtaining quantitative data without expensive or highly specialized equipment. Key features • Ideal for root tissues of similar diameter. • Few preparation steps, and only simple lab equipment is needed. • Data can be obtained in three days.