Home › Botany › Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter
Botany JoVE (Open Access) Citable · DOI

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

DOI: 10.3791/54586-v
What you'll learn
  • ✓ Isolate and transfect Arabidopsis protoplasts with luminescent circadian reporters
  • ✓ Perform real-time luminescent imaging to measure circadian clock phenotypes
  • ✓ Analyze circadian rhythm data to identify mutant clock defects rapidly
Protocol

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

Difficulty
advanced
Total time
~4–6 hours per genotype (protoplast isolation through imaging acquisition)
Model organism
Arabidopsis thaliana
Biosafety
BSL-1

Steps

1
Isolate protoplasts from Arabidopsis leaf tissue

Enzymatically digest cell walls to generate viable protoplasts from seedling leaves. This step yields the single-cell substrate for transfection and circadian reporter assay.

▶ 00:39
2
Transfect protoplasts with luminescent clock reporter construct

Introduce circadian reporter plasmids (e.g., LUC fusion) into isolated protoplasts via PEG-mediated transfection. Transient expression enables rapid phenotypic screening without stable transformation.

▶ 03:16
3
Image and quantify luminescence from transfected protoplasts

Set up luminometric imaging in darkness and record bioluminescence over ~24–48 hours. Extract raw signal traces for each protoplast or population to assess circadian periodicity and amplitude.

▶ 05:03
4
Analyze circadian phenotypes from luminescence time-series data

Apply Fourier or periodogram analysis to determine period, phase, and damping of circadian oscillations. Compare wild-type and mutant protoplast rhythms to identify altered clock function.

▶ 05:48
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