Keywords

CDPK, Calcium signaling, Ca2+ decoding, FRET, ABA, Stomatal closure, SLAC1, Flg22, Conformational sensors

Reference

DOI: 10.1093/plcell/koad159

Abstract

This study introduces CPKaleon, a genetically encoded FRET-based reporter, to visualize real-time conformational changes of Arabidopsis calcium-dependent protein kinases (CDPKs/CPKs) in living cells.
Focusing on highly Ca2+-sensitive CPK21 and less Ca2+-sensitive CPK23, the authors tracked how CDPKs respond to cytosolic calcium oscillations.

  • CPK21-FRET sensors, but not CPK23, mirrored oscillatory Ca2+ patterns in tobacco pollen tubes and Arabidopsis guard cells, establishing CPK21 as a real-time decoder of calcium signatures.
  • Upon ABA and flg22 treatments, CPK21 conformation shifted in response to Ca2+, indicating its role in decoding stimulus-specific Ca2+ signatures to regulate SLAC1/SLAH3-mediated stomatal closure.
  • The study highlights isoform-specific Ca2+ decoding capacities among CDPKs, with implications for plant stress and immune signaling.

Notes

1. Experimental Approach: Development of CDPK-FRET Reporters

  • Genetically encoded FRET biosensor (CPKaleon) created to detect Ca2+-dependent conformational changes in CDPKs.
  • Focus on two functionally related but differentially sensitive CDPKs:
    • AtCPK21: high Ca2+-sensitivity.
    • AtCPK23: low Ca2+-sensitivity.
  • Kinase-deficient variants used to ensure FRET changes reflect conformation, not phosphorylation.
  • Used tobacco pollen tubes and Arabidopsis guard cells for in vivo imaging.
  • RasMol employed to calculate intramolecular distances for FRET design.

Smart FRET design allows direct monitoring of CDPK conformational change, an elegant solution to visualize real-time signaling!

2. Key Findings

  • CPK21, but not CPK23, mirrors cytosolic Ca2+ oscillations in pollen tubes, showing isoform-specific sensitivity and reversibility.
  • Both ABA and flg22 induce CPK21 conformational changes, tightly linked to Ca2+ elevation.
  • Ca2+-dependent conformational shifts in CPK21 precede functional responses like SLAC1 activation, establishing a link between Ca2+ decoding and stomatal closure.
  • CPK23’s low responsiveness to Ca2+ may stem from its Ser362 (instead of Ile) at position 31 of PS helix—confirmed by mutagenesis:
    • CPK21 Ile373Ser mutation reduced Ca2+-sensitivity.
    • CPK23 Ser362Ile increased Ca2+-responsiveness.
  • Ca2+ selectivity confirmed: Increasing [Mg2+] reduced FRET signal in absence of Ca2+, demonstrating EF hands’ Ca2+ specificity.
  • Time delay of ~8 seconds observed between Ca2+ peaks and CPK21-FRET response, especially for the first spike, suggesting fine-tuned activation dynamics.

Conformation monitoring highlights that CDPKs are not just Ca2+ binders but true signal integrators!

3. Functional and Conceptual Implications

  • CDPK21 functions as a Ca2+ decoder — linking distinct Ca2+ signatures to specific outputs like stomatal closure.
  • Distinct Ca2+-sensitivity between CPK21 and CPK23 allows fine-tuning of responses under various stress conditions (e.g., ABA vs. flg22).
  • Discovery of Ca2+-independent phosphorylation on CPK23 (S362) and its impact on sensitivity offers a novel layer of regulation in CDPK activation.
  • Suggests stimulus-specific Ca2+ signatures are “read” only by the right CDPK isoforms — providing specificity in signaling networks with shared Ca2+ fluxes.

Isoform-specific Ca2+ decoding allows one signal (Ca2+) to achieve multiple, specific outcomes!

4. RD’s Takeaways and Reflections

  • LOVE the use of conformational FRET to track Ca2+ decoding in live cells — elegant, dynamic, and adds spatial context.
  • Raises exciting possibilities:
    • Could similar conformational sensors be built for other kinases (e.g., animal CaMKII)?
    • What governs CDPK conformational shift: Ca2+ alone or interplay with auto-phosphorylation? (note: they used kinase-dead variants, but in real life, P may shape the conformation too).
  • The ~8s delay between Ca2+ and CPK21 shifts is super intriguing — biological filtering mechanism? Threshold setting?
  • Their mention of stimulus-specific “Ca2+ signatures” and spatial organization of Ca2+ channels = hints toward spatial code + frequency code in signaling — aligns well with neural systems thinking.
  • Great system to explore how CDPKs act as plant “frequency decoders”, akin to CaMKII in neurons — need a CaMKII vs CDPK comparative piece!

This study is a milestone — first in planta visualization of CDPK decoding in real time, showing these kinases are dynamic, nuanced decoders of Ca2+ information, not simple switches.

Take-Home Messages

  • CDPK21 dynamically decodes cytosolic calcium transients in response to ABA and flg22, linking calcium signaling to stomatal control.
  • CDPK-FRET sensors (CPKaleon) provide a powerful real-time tool to monitor kinase conformational changes in live plants.
  • Isoform-specific Ca2+ sensitivity (e.g., CPK21 vs. CPK23) adds fine-tuning to plant calcium signaling networks.
  • Post-translational modifications (like S362 in CPK23) adjust calcium responsiveness, introducing new regulatory layers.
  • Real-time conformational imaging transforms our understanding of how calcium signals are decoded at the protein level.

Final Reflection

“If CaMKII is the neuronal frequency decoder, CDPKs are the plant equivalent — parsing calcium signatures into precise stress responses. This paper shows that, for the first time, we can watch this decoding process unfold in real time, inside living cells.”