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

Cytosolic Ca^(2+)plays a key role in signal transduction in plants.Calcium imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca^(2+)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 Ca^(2+)fluorescent probes successfully entered the cytoplasm but did not enter the vacuole.Both the Ca^(2+)chelator(EGTA)and Ca^(2+)channel blocker(La3+)reduced the fluorescence intensity in the cytoplasm.The calcium ionophore A23187 increased the fluorescence intensity in the cytoplasm at 5μmol/L but decreased it at 50μmol/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 Ca^(2+)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 5μmol/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.