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Nuclear RNA quantification in protoplast cell-cycle phases.

C Bergounioux¹, C Perennes, S C Brown

¹Laboratoire de Physiologie Végétale Moléculaire, Université d'Orsay, Faculté des Sciences, France.

|January 1, 1988

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This study demonstrates that RNA synthesis is crucial for plant cell DNA replication. Increased RNA levels signal cells to initiate DNA replication from the G1 phase.

Area of Science:

Plant cell biology
Molecular biology
Cytometry

Background:

Acridine orange staining and flow cytometry are established methods for measuring cellular DNA and RNA levels.
Applying these techniques to plant protoplasts requires validation for plant-specific biological contexts.

Purpose of the Study:

To validate the use of acridine orange staining and flow cytometry for quantifying DNA and RNA in plant protoplast nuclei.
To investigate the role of RNA synthesis in the cell cycle progression of plant protoplasts.

Main Methods:

Isolation of nuclei from cultured plant protoplasts.
Acridine orange staining for DNA and RNA quantification via flow cytometry.
Treatment with ribonuclease, ribonuclease inhibitor, and actinomycin D to validate assay specificity and assess RNA synthesis.

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Main Results:

The acridine orange method was validated for plant material, with ribonuclease treatment reducing RNA staining but not DNA histograms.
RNA synthesis was detected in Petunia hybrida protoplasts starting at 18 hours in culture.
A critical increase in RNA levels was necessary for cells to transition from G1 to DNA replication (G1B phase).
G2 phase nuclei exhibited an RNA:DNA ratio comparable to G1 phase nuclei.

Conclusions:

Acridine orange staining coupled with flow cytometry is a reliable method for assessing DNA and RNA content in plant protoplast nuclei.
RNA synthesis plays a critical regulatory role in initiating DNA replication in plant cells.
The G1B phase represents a critical checkpoint regulated by RNA levels before DNA replication commences.