Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica

Lina Qiu,Yongzhang Wang,Haiyong Qu

doi:10.1038/s41438-020-0315-3

Lina Qiu, Yongzhang Wang + Show 1 more

Open Access

https://doi.org/10.1038/s41438-020-0315-3

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Journal: Horticulture Research	Publication Date: Jun 1, 2020
Citations: 18	License type: open-access

Affiliation: Qingdao Agricultural University

Abstract

Cytosolic Ca2+ plays a key role in signal transduction in plants. Calcium imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca2+ content. Here, we used mature ‘Fuji’ apples (Malus pumila Mill.) to obtain viable protoplasts from flesh tissue cells by enzymatic hydrolysis; then, three small-molecule fluorescent probes (fluo-8/AM, fluo-4/AM, and rhod-2/AM) were loaded into the protoplasts. All three Ca2+ fluorescent probes successfully entered the cytoplasm but did not enter the vacuole. Both the Ca2+ chelator (EGTA) and Ca2+ channel blocker (La3+) reduced the fluorescence intensity in the cytoplasm. The calcium ionophore A23187 increased the fluorescence intensity in the cytoplasm at 5 µmol/L but decreased it at 50 µmol/L. Additionally, A23187 reversed the fluorescence intensity in the cytoplasm, which was decreased by La3+. Ionomycin is also a calcium ionophore that can increase the fluorescence intensity of calcium in the cytoplasm. These results suggest that small-molecule Ca2+ fluorescent probes can be used to detect changes in cytosolic calcium levels in the cells of fruit flesh tissue. In addition, the optimum concentration of fluo-8/AM was determined to be 5 µmol/L. This was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue cells. This study provides a new method to study calcium signal transduction in fruit flesh tissue.

Highlights

Calcium influences many fruit qualities[1,2]; it affects fruit firmness, sugar content, storage period, and physiological disorders during storage[3,4]
The optimum concentration of fluo-8/AM was determined to be 5 μmol/L. This was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue cells
As there is a large vacuole in the center of the flesh cell with the cytoplasm squeezed around the cell (Supplementary Fig. S1A,B), fluorescence appeared around the cell

Summary

Introduction

Calcium influences many fruit qualities[1,2]; it affects fruit firmness, sugar content, storage period, and physiological disorders during storage[3,4]. Fluorescent probes can be used to detect changes in cytosolic calcium levels in the cells of fruit flesh tissue. This was the first time that protoplasts have been isolated from apple flesh tissue cells and small-molecule fluorescent probes have been used to detect calcium in the cytoplasm of flesh tissue cells.

Results

Conclusion