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.

The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca²⁺ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca²⁺ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs.

Apple bitter pit primarily occurs during fruit ripening and storage; however, its formation mechanism remains unclear. Although it is considered that Ca²⁺deficiency causes metabolic disorders in apples, there have been few studies on the mechanism of the bitter pit from the perspective of cell structure. At the fruit ripening stage, the fruit with a bitter pit on the tree was taken as the research material. In this study, the microscopic observation revealed numerous amyloplasts in the pulp cells of apples affected with bitter pit, but not in the healthy pulp. Furthermore, the results of fluorescence staining and transmission electron microscopy (TEM) revealed that the bitter pit pulp cells undergo programmed cell death (PCD), their nuclear chromosomes condense, and amyloplast forms autophagy. The cytoplasmic Ca²⁺concentration in the healthy fruits was lowest near the peduncle, followed by that in the calyx, whereas it was highest at the equator. In contrast, the cytoplasmic Ca²⁺concentration in apple fruits showing bitter pit disorder was lowest near the peduncle and highest in the calyx. Moreover, the cytosolic Ca²⁺concentration in the flesh cells of apples with the bitter pit was much lower than that in the healthy apple flesh cells; however, the concentration of Ca²⁺in the vacuoles of fruits with the bitter pit was higher than that in the vacuoles of healthy fruits. In summary, bitter pit pulp cells contain a large number of amyloplasts, which disrupts the distribution of Ca²⁺in the pulp cells and causes PCD. These two processes lead to an imbalance in cell metabolism and induce the formation of a bitter pit.