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

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/F 0 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.

Plant phenotyping relevance

植物体内のグルコースを定量する遺伝子コード型蛍光センサーを開発・最適化し、シロイヌナズナ根で検証しているため、植物生理状態の取得法が中心です。

Code and data availability

植物フェノタイピング解析を再現する公開資産であることを、入力本文と直接リンクから確認できなかったため保留しました。

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