Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta

Abstract

Abstract Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

Plant phenotyping relevance

植物体内のCa2+依存的な生理状態をリアルタイム可視化するFRETレポーターを開発しており、植物表現型取得法が研究の中心である。

Code and data availability

The supplied blocks describe FRET-based CDPK conformational imaging in pollen tubes and guard cells, but contain no public phenotype/trait datasets, plant imaging data deposits, or author analysis code with an explicit public URL. The Addgene plasmid (pAGM9121) is a generic MoClo cloning vector, not a paper-specific ph

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