Determination of protoplast growth properties using quantitative single-cell tracking analysis

Area of Science:

• Plant biology
• Cell biology
• Biotechnology

Background:

• Quantitative single-cell analysis is common in animals but limited in plants.
• Plant single-cell analysis using tobacco leaf protoplasts was explored.
• Protoplasts, cell-wall free plant cells, can regenerate cell walls and proliferate.

Purpose of the Study:

• To develop and apply high-throughput microscopic image analysis for plant single-cell studies.
• To quantify developmental properties of tobacco protoplasts during early cultivation.
• To compare wild-type tobacco cells with those expressing the antiapoptotic protein AtBAG4.

Main Methods:

• High-throughput automated microscopy and image processing pipelines were used.
• Protoplast development was quantified in multi-well plates.
• Growth parameters of wild-type and AtBAG4-expressing cells were compared.

Main Results:

• AtBAG4-expressing protoplasts exhibited a higher percentage of cells with increased area.
• These cells showed significantly increased growth and proliferation rates.
• Single-cell expansion suggested a BAG4-mediated increase in potassium channel abundance.

Conclusions:

• AtBAG4 expression enhances plant single-cell growth and proliferation.
• These findings align with AtBAG4's known roles in stress resilience and survival.
• The developed pipeline enables novel biotechnological screening for improved plant properties via single-cell analysis.