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

Abstract

Summary 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/F 0 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.

Plant phenotyping relevance

植物内グルコース動態を測定する遺伝子 encoded センサーの開発・感度評価・シロイヌナズナ根での実証が中心であり、植物の生理状態を取得するフェノタイピング手法に該当する。

Code and data availability

No public paper-specific phenotyping asset is available. The raw data are stated to be deposited as an ARC 'under doi (available for publication)', i.e., not yet publicly released and without a URL. Addgene plasmids are biological reagents, not phenotype datasets, images, analysis code, or models. The MGlucoMeter1.0 HH

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