Fluorescence-Activated Cell Sorting Using the D-Root Device and Optimization for Scarce and/or Non-Accessible Root...

Area of Science:

• Plant Biology
• Molecular Biology
• Genomics

Background:

• Fluorescence-activated cell sorting (FACS) is crucial for isolating specific cell populations for transcriptomic analysis.
• Existing protoplast extraction protocols are unsuitable for root cells grown in darkness, a non-natural condition.
• Root cells typically grow in darkness, and current methods fail to capture this under natural conditions.

Purpose of the Study:

• To establish an optimized Fluorescence-Activated Cell Sorting (FACS) protocol for isolating root protoplasts from plants grown in darkness.
• To enable transcriptomic analysis of specific, even scarce, root cell types under simulated natural conditions.
• To provide a robust method for studying root cell-type specific gene expression.

Main Methods:

• Utilized Arabidopsis green fluorescent protein (GFP)-marked lines and the D-Root device to grow roots in darkness.
• Optimized protoplast isolation using a minimal enzyme concentration for efficient yield.
• Employed Fluorescence-Activated Cell Sorting (FACS) with mCherry fluorescent protein for purification of specific cell types.

Main Results:

• Successfully isolated viable root protoplasts from Arabidopsis grown in darkness using an optimized FACS protocol.
• Demonstrated the protocol's effectiveness in isolating scarce cell types from differentiated tissues.
• Generated cDNA and sequencing libraries from isolated RNA, confirming suitability for genome-wide transcriptomic analysis.

Conclusions:

• The developed FACS protocol effectively isolates root protoplasts from plants grown in darkness.
• This method allows for cell-type specific transcriptomic profiling of root cells under near-natural conditions.
• The protocol is versatile and applicable to various Arabidopsis lines and cell types for gene expression studies.