Establishment of genetically encoded biosensors for cytosolic boric acid in plant cells

Abstract

SUMMARY Boron (B) is an essential micronutrient for plants. To maintain B concentration in tissues at appropriate levels, plants use boric acid channels belonging to the NIP subfamily of aquaporins and BOR borate exporters. To regulate B transport, these transporters exhibit different cell‐type specific expression, polar localization, and B‐dependent post‐transcriptional regulation. Here, we describe the development of genetically encoded biosensors for cytosolic boric acid to visualize the spatial distribution and temporal dynamics of B in plant tissues. The biosensors were designed based on the function of the NIP 5 ; 1 5′‐untranslated region ( UTR ), which promotes mRNA degradation in response to an elevated cytosolic boric acid concentration. The signal intensities of the biosensor coupled with Venus fluorescent protein and a nuclear localization signal ( uNIP 5 ; 1‐Venus ) showed negative correlation with intracellular B concentrations in cultured tobacco BY ‐2 cells. When expressed in Arabidopsis thaliana , uNIP 5 ; 1‐Venus enabled the quantification of B distribution in roots at single‐cell resolution. In mature roots, cytosolic B levels in stele were maintained under low B supply, while those in epidermal, cortical, and endodermal cells were influenced by external B concentrations. Another biosensor coupled with a luciferase protein fused to a destabilization PEST sequence ( uNIP 5 ; 1‐Luc ) was used to visualize changes in cytosolic boric acid concentrations. Thus, uNIP 5 ; 1‐Venus / Luc enables visualization of B transport in various plant cells/tissues.

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