Evidence for rapid hydrolysis of shoot-derived sucrose using an ultrasensitive ratiometric Matryoshka-type MGlucoMeter sensor

To enable sensitive in vivo monitoring of the glucose transport and metabolism, we developed a series of ultrasensitive and ratiometric genetically encoded nanosensors by inserting a Matryoshka dual fluorophore cassette consisting of cpsfGFP and LSSmApple into the glucose binding protein ttGBP from Thermus thermophilus. The initial MGlucoMeter1.0 was subjected to an alanine scan of the hinge region producing 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 1.1-216 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, MGlucoMeter2.6-15μ, MGlucoMeter2.6-700μ, MGlucoMeter2.6-1m, and MGlucoMeter2.6-7m cover a detection range between 40 nM - 55 mM. When expressed from a ubiquitous promoter in the cytosol of the Arabidopsis gene silencing mutant rdr6, MGlucoMeter2.6-1m reports time- and concentration-dependent accumulation of glucose in seedling roots. The sensor also detects rapid hydrolysis of shoot-derived sucrose in the root tip.