# Evidence for rapid hydrolysis of shoot‐derived sucrose using an ultrasensitive ratiometric matryoshka‐type MGlucoMeter sensor

## TL;DR

A new ultra-sensitive sensor called MGlucoMeter was developed to track glucose levels in plants, revealing rapid sugar breakdown in roots.

## Contribution

MGlucoMeter is a novel ratiometric sensor with ultrasensitivity for in vivo glucose monitoring across a wide dynamic range.

## Key findings

- MGlucoMeter detected rapid hydrolysis of shoot-derived sucrose in root tips.
- The sensor reports time- and concentration-dependent glucose accumulation in Arabidopsis seedling roots.
- MGlucoMeter variants cover a detection range from ~40 nm to 55 mm glucose.

## Abstract

To enable sensitive in vivo monitoring of the glucose transport and metabolism, we developed a series of ultrasensitive and ratiometric genetically encoded sensors (MGlucoMeter) by inserting a Matryoshka dual fluorophore cassette consisting of cpsfGFP (circularly permuted superfolder GFP) and LSSmApple (Large Stokes Shift mApple) into the glucose‐binding protein ttGBP (Thermus thermophilus glucose‐binding protein) from Thermus thermophilus. The initial MGlucoMeter1.0 version was subjected to an alanine scan of the hinge region producing the more sensitive MGlucoMeter2.6 with a glucose‐induced ΔF/F0 change of 3.0, an affinity for glucose of 15 μm, and an approximate detection range of 1–215 μm. To generate variants suitable for in vivo measurements, a series of affinity mutants was generated by mutating two histidines predicted to be involved in substrate binding. MGlucoMeter2.6‐353n (affinity 353 nm), MGlucoMeter2.6‐15 μ (affinity 15 μm), MGlucoMeter2.6‐700 μ (affinity 700 μm), MGlucoMeter2.6‐1 m (affinity 1 mm), and MGlucoMeter2.6‐7 m (affinity 7 mm) cover a combined detection range between ∼40 nm–55 mm. When expressed from a ubiquitous promoter in the cytosol of the Arabidopsis gene‐silencing mutant rdr6 (RNA‐dependent RNA polymerase 6), MGlucoMeter2.6‐1 m reports time‐ and concentration‐dependent accumulation of glucose in seedling roots after external addition of glucose. The sensor also detected rapid release of sugars in the root tip and rapid hydrolysis of the shoot‐derived sucrose.

MGlucoMeter, a high‐sensitivity ratiometric genetically encoded sensor, can report changes in glucose levels in live cells and organisms. Here, MGlucoMeter detected the hydrolysis of leaf‐derived sucrose in the root tip unloading zone.