Visualising Apoplastic Fluid Flow and pH Dynamics in Arabidopsis Roots

Original video: https://youtu.be/LGUoDQ6l1BA

This video combines time-lapse bioluminescence imaging and pH sensing to investigate apoplastic fluid flow and pH dynamics in Arabidopsis roots. Using the genetically encoded indicator 35S::Cel1SP-GeNL.

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 pKa 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. 35S::Cel1SP-GeNL is the name of a genetically modified line of A. thaliana plants used in this research. The researchers fused the coding sequence for a pH-sensitive bioluminescent protein called GeNL with a signal peptide from a cellulase enzyme (Cel1). This fusion protein, Cel1SP-GeNL, was then introduced into A. thaliana plants using the CaMV 35S promoter, which drives expression throughout the plant. The goal was to localize GeNL in the apoplast, the space outside the plasma membrane, to measure the pH of the apoplastic fluid.

Keywords: Bioluminescence, pH homoeostasis, pH indicator, Root imaging, Apoplast, Time-lapse

Citation:

Tran, Q., Osabe, K., Entani, T., Wazawa, T., Hattori, M., & Nagai, T. (2022). Application of Green-enhanced Nano-lantern as a bioluminescent ratiometric indicator for measurement of Arabidopsis thaliana root apoplastic fluid pH. Plant, Cell & Environment, 45(10), 3157–3170. https://doi.org/10.1111/pce.14404

‌Published on: 21 July 2022

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