staining-the-cytoplasmic-ca2-with-fluo-4am-in-apple-pulp

Staining the Cytoplasmic Ca<sup>2+</sup> with Fluo-4/AM in Apple Pulp

Cytosolic Ca2+ plays a key role in plant development. Calcium imaging is the most versatile method to detect dynamic changes in Ca2+ in the cytoplasm. In ...

Qingdao Agricultural University

Cytosolic Ca plays a key role in signal transduction in plants. Calcium imaging is the most common approach to studying dynamic changes in the cytoplasmic Ca content. Here, we used mature 'Fuji' apples (.) 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 fluorescent probes successfully entered the cytoplasm but did not enter the vacuole. Both the Ca chelator (EGTA) and Ca channel blocker (La) 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 La. Ionomycin is also a calcium ionophore that can increase the fluorescence intensity of calcium in the cytoplasm. These results suggest that small-molecule Ca 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.

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

Drought is a major environmental factor that significantly limits crop yield and quality worldwide. Basic helix-loop-helix (bHLH) transcription factors have been reported to participate in the regulation of various abiotic stresses. In this study, a bHLH transcription factor in apple, , which contains a highly conserved bHLH domain, was isolated and characterized. qRT-PCR and  analyses showed that  was notably induced in response to dehydration stress. Compared with the wild-type (WT), transgenic apple calli overexpressing  displayed greater resistance to PEG6000 treatment. In contrast, the  lines were more sensitive to PEG6000 treatment than WT. Moreover, ectopic expression of  in tobacco improved tolerance to water deficit stress, and plants exhibited higher germination rates and survival rates, longer roots, and lower ABA-induced stomatal closure and leaf water loss than the WT control. Furthermore, overexpression of  in tobacco also led to lower electrolyte leakage, malondialdehyde contents, and reactive oxygen species (ROS) accumulation and upregulation of the expression of some ROS-scavenging and stress-responsive genes under water deficit stress. In addition,  transgenic tobacco plants exhibited improved tolerance to oxidative stress compared with WT. In conclusion, these results indicate that  acts as a positive regulator of water stress responses through modulating stomatal closure and ROS-scavenging in tobacco.

, an Apple bHLH Transcription Factor, Confers Water Stress Resistance by Regulating Stomatal Closure and ROS Homeostasis in Transgenic Tobacco.

Apple bitter pit primarily occurs during fruit ripening and storage; however, its formation mechanism remains unclear. Although it is considered that Ca 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 Ca 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 Ca concentration in apple fruits showing bitter pit disorder was lowest near the peduncle and highest in the calyx. Moreover, the cytosolic Ca 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 Ca 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 Ca 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.

Article	Year
Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of . Horticulture research\| PubMed ID: 32528703	2020
, an Apple bHLH Transcription Factor, Confers Water Stress Resistance by Regulating Stomatal Closure and ROS Homeostasis in Transgenic Tobacco. Frontiers in plant science\| PubMed ID: 33163009	2020
Accumulation of Abnormal Amyloplasts in Pulp Cells Induces Bitter Pit in . Frontiers in plant science\| PubMed ID: 34630490	2021

Lina Qiu

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