Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp

Area of Science:

• Plant Biology
• Cell Physiology

Background:

• Cytosolic calcium (Ca2+) is crucial for plant development.
• Calcium imaging is vital for observing dynamic Ca2+ changes in the cytoplasm.

Purpose of the Study:

• To develop a method for isolating viable protoplasts from fruit pulp cells.
• To detect and visualize cytosolic Ca2+ in fruit pulp cells using a fluorescent reagent.

Main Methods:

• Enzymatic hydrolysis was used to obtain viable protoplasts from fruit pulp cells.
• Isolated protoplasts were incubated with Fluo-4/AM, a small-molecule fluorescent reagent.
• Calcium channel blocker La3+ was used to validate the specificity of the fluorescent probe.

Main Results:

• Fluo-4/AM successfully stained cytosolic Ca2+ in fruit pulp protoplasts without accumulating in vacuoles.
• Incubation with La3+ led to a decrease in cytoplasmic fluorescence intensity, confirming Ca2+ detection.
• The developed method allows for effective isolation and Ca2+ detection in fruit flesh cells.

Conclusions:

• Fluo-4/AM is a suitable reagent for detecting cytosolic Ca2+ changes in fruit pulp cells.
• The presented method provides a reliable approach for studying Ca2+ dynamics in fruit flesh protoplasts.