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

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. 

Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs.

Hard end is a physiological disorder of pear fruit that is frequently observed in the 'Whangkeumbae' (Pyrus pyrifolia) variety, however, the mechanisms that are involved in its development are poorly understood. In this study, we explored the causes of hard end disorder in pear fruit in relation to calcium deficiency. During fruit development, the ratio of Ca/N, Ca/K, Ca/Mg and the content of B were significantly lower in the hard end fruit as compared to normal fruit. However, no calcium deficiency was detected in the soil and leaves of the orchard where the hard end fruit were located. Additionally, the Ca influx in the calyx of hard end fruit was lower than that of normal fruit at 90 d after anthesis. The free Ca and storage Ca in the flesh cells of hard end fruit were less than that of normal fruit during fruit development, while an opposite tendency was observed at 120 d after anthesis (harvest day). In hard end fruit, the Ca transport-related gene, PpCNGC1 (Cyclic nucleotide-gated ion channel 1), was up-regulated; whereas the Ca sensor-related genes of PpCIPKs, PpCDPK28 and PpCML41 were all down-regulated. Spraying with a 2% calcium chloride (CaCl) solution inhibited the incidence rate of hard end disorder and decreased fruit firmness and lignin content during storage. Additionally, the ratio of Ca/N, Ca/K, Ca/Mg and the content of B all increased on harvest day. Our study suggests that low Ca influx leads to less Ca into the pear fruit, which results in an intracellular imbalance of Ca and consequently triggers the development of hard end disorder.

The changes of intracellular calcium concentration and distribution in the hard end pear (Pyrus pyrifolia cv. 'Whangkeumbae') fruit.

At the early stage of pollination, the difference in gene expression between compatibility and incompatibility is highly significant about the pollen-specific expression of the LRR gene, resistance, and defensin genes. In Rosaceae, incompatible pollen can penetrate into the style during the gametophytic self-incompatibility response. It is therefore considered a stylar event rather than a stigmatic event. In this study, we explored the differences in gene expression between compatibility and incompatibility in the early stage of pollination. The self-compatible pear variety "Jinzhuili" is a naturally occurring bud mutant from "Yali", a leading Chinese native cultivar exhibiting typical gametophytic self-incompatibility. We collected the styles of 'Yali' and 'Jinzhuili' at 0.5 and 2 h after self-pollination and then performed high-throughput sequencing. According to the KEGG analysis of the differentially expressed genes, several metabolic pathways, such as "Plant hormone signal transduction", "Plant-pathogen interaction", are the main pathways was the most represented pathway. Quantitative PCR was used to validate these differential genes. The expression levels of genes related to pollen growth and disease inhibition, such as LRR (Leucine-rich repeat extensin), resistance, defensin, and auxin, differed significantly between compatible and incompatible pollination. Interestingly, at 0.5 h, most of these genes were upregulated in the compatible pollination system compared with the incompatible pollination system. Calcium transport, which requires ATPase, also demonstrated upregulated expression. In summary, the self-incompatibility reaction was initiated when the pollen land on the stigma.

RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination.

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 .

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.

Accumulation of Abnormal Amyloplasts in Pulp Cells Induces Bitter Pit in .

The pollen tube is ideal for studying cell polar growth, and observing the ultrastructure of the pollen tube tip using transmission electron microscopy (TEM) is the primary method for studying pollen tube growth. The preparation of ultrathin sections of the pollen tube tip sample is important for its successful microscopic observation. The direction of pollen tube growth in vitro is irregular, and it is difficult to dissect the tip of the pollen tube during ultrathin sectioning. Here, we used two methods to efficiently obtain an ultrathin section of the pollen tube tip of Pyrus. In the first method, laser micro-cutting was used to obtain the pollen tube tip, followed by ultrathin sectioning. In the other method, the pollen tubes were cultured in the same growth direction, followed by ultrathin sectioning. Ultrathin sections, which were observed via TEM, showed typical characteristics of the pollen tube tip, such as dense vesicles, numerous mitochondria, and secretory vesicles of the Golgi. We concluded that these two methods are effective in pollen tube tip sample preparation for ultrathin sectioning and provide the foundation for observing the ultrastructure of pollen tube tips.

Haiyong Qu

College of Horticulture

Qingdao Agricultural University

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

Haiyong Qu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Horticulture

Qingdao Agricultural University

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

College of Horticulture

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