Application of Green‐enhanced Nano‐lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH

Plant root absorbs water and nutrients from the soil, and the root apoplastic fluid (AF) is an important intermediate between cells and the surrounding environment. The acid growth theory suggests that an acidic AF is needed for cell wall expansion during root growth. However, technical limitations have precluded the quantification of root apoplastic fluid pH (AF‐pH). Here, we used Green‐enhanced Nano‐lantern (GeNL), a chimeric protein of the luciferase NanoLuc (Nluc) and the green fluorescent protein mNeonGreen (mNG), as a ratiometric pH indicator based on the pH dependency of bioluminescence resonance energy transfer efficiency from Nluc to mNG. Luminescence spectrum of GeNL changed reciprocally from pH 4.5 to 7.5, with a pK (a) of 5.5. By fusing GeNL to a novel signal peptide from Arabidopsis thaliana Cellulase 1, we localised GeNL in A. thaliana AF. We visualised AF dynamics at subcellular resolution over 30 min and determined flow velocity in the maturation zone to be 0.97± 0.06 μm/s. We confirmed that the developing root AF is acidic in the pH range of 5.1−5.7, suggesting that the AF‐pH is tightly regulated during root elongation. These results support the acid growth theory and provide evidence for AF‐pH maintenance despite changes in ambient pH.