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An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca²⁺ and H⁺ reveals new insights into ion signaling in plants.

ABSTRACT: Whereas the role of calcium ions (Ca²⁺ ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca²⁺ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca²⁺ - and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca²⁺ -dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca²⁺ . In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca²⁺ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca²⁺ -increases and cytosolic acidification by c. 2 min, suggesting a Ca²⁺ /pH-independent early pathogen signaling step. Imaging Ca²⁺ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca²⁺ - and pH-responses. We propose close interrelation in Ca²⁺ - and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.

SUBMITTER: Li K

PROVIDER: S-EPMC8383442 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

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An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca²⁺ and H⁺ reveals new insights into ion signaling in plants.

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Dataset Information

An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca2+ and H+ reveals new insights into ion signaling in plants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca²⁺ and H⁺ reveals new insights into ion signaling in plants.