Evidence for rapid hydrolysis of shoot-derived sucrose using an ultrasensitive ratiometric matryoshka-type...

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.