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

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.

In S-RNase-based self-incompatibility, S-RNase was previously thought to function as a selective RNase that inhibits pollen whose S-haplotype matches that in the pistil. In this study, we showed that S-RNase has a distinct effect on the regulation of Ca-permeable channel activity in the apical pollen tube in Pyrus pyrifolia. While non-self S-RNase has no effect, self S-RNase decreases the activity of Ca channels and disrupts the Ca gradient at the tip of the growing pollen tube during the gametophytic self-incompatibility (GSI) response. Extracellular Ca influx was suppressed 5min after self S-RNase treatment, and self-pollen tube growth was reduced at 50min after self S-RNase treatment. In the self-incompatible response, the expression of Ca-related genes was inhibited before RNA degradation. Therefore, self S-RNase suppresses Ca influx prior to arresting pollen tube growth via RNA degradation.

The role of Ca and Ca channels in the gametophytic self-incompatibility of Pyrus pyrifolia.

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.

Compost amendment reportedly improved apple tree growth in replant soils. However, its effects should be evaluated at different soil depths and locations. This study investigated the impact of soil improvement with compost on soil physicochemical properties and bacterial community structure of a replanted apple orchard in comparison with the original orchard without compost improvement. The V1-V3 region of the bacterial 16S rRNA gene was subjected to high-throughput 454 pyrosequencing, and data were analyzed using the Mothur pipeline. The results showed that the soil improvement benefited tree growth and fruit quality during the study period. The compost amendment markedly increased tree height and stem diameter by a range of 6.1%-21.0% and 4.0%-14.0%, respectively. Fruit yield (9.5%), average weight (9.6%), and soluble solid content (5.6%) were also increased by compost amendment compared to those of the unimproved treatment. The pH, organic matter, and available N, P, and K contents were significantly increased by 5.7%-21.9%, 0.2%-62.9%, 9.3%-29.3%, 36.7%-64.5%, and 17.2%-100.3% in the compost improved soil. The pyrosequencing data showed that the soil improvement changed the bacterial community structure at all soil depths (0-20 cm and 20-40 cm) and locations (in-row and inter-row) considered; e.g., the relative abundance of Proteobacteria (20.2%), Bacteroidetes (2.5%), and Cyanobacteria (1.0%) was increased while that of Chloroflexi (5.5%), Acidobacteria (5.2%), Nitrospirae (4.5%), Gemmatimonadetes (3.8%), and Actinobacteria (1.8%) was decreased. The relative abundance of some dominant genera Burkholderia (2.3%), Pseudomonas (1.0%), and Paenibacillus (0.5%) were enhanced in the compost improved soil. Moreover, other dominant genera such as Nitrospira (6.4%), Gemmatimonas (2.2%), and Phenylobacterium (0.3%) were reduced by the application of compost. Our results indicate that soil improvement benefits the growth of tree and fruit quality, and is likely mediated by increased soil pH, organic matter, and available nutrient contents and beneficial bacterial community composition.

Soil amendment alters soil physicochemical properties and bacterial community structure of a replanted apple orchard.

The plant-specific TCP transcription factors play different functions in multiple processes of plant growth and development. TCP family genes have been identified in several plant species, but no comprehensive analysis of the TCP family in grapevine has been undertaken to date, especially their roles in fruit development.

Genome-wide identification and transcript analysis of TCP transcription factors in grapevine.

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.

The B-BOX (BBX) proteins are the class of zinc-finger transcription factors and can regulate plant growth, development, and endure stress response. In plants, the BBX gene family has been identified in Arabidopsis, rice, and tomato. However, no systematic analysis of BBX genes has been undertaken in grapevine.

Genome-wide identification and analysis of B-BOX gene family in grapevine reveal its potential functions in berry development.

The color of apple skin, particularly anthocyanin-based coloration, is a key factor determining market acceptance. The mechanisms of anthocyanin accumulation in apples with different skin color patterns (i.e., striped and blushed) were analyzed. In total, 14 anthocyanins and 5 procyanidins were simultaneously assayed in red blushed-skin mutants (CF-B1 and CF-B2) and red striped-skin parents (CF-S1 and CF-S2), and 13 significant differences were revealed. Anthocyanin accumulation was significantly higher in the red blushed-skin apples than it was in the parents. The transcript levels of anthocyanin biosynthesis genes and regulatory factors (, , and ) were associated with different skin color patterns during the coloring period at 4, 6, and 8 days after the fruits were debagged. The methylation levels of the  promoter regions -1203 to -779 bp, -1667 to -1180 bp, and -2295 to -1929 bp were associated with different skin color patterns, and there was more methylation in red striped-skin apples. These results improve our understanding of anthocyanin accumulation and its underlying molecular mechanism in apples with different skin color patterns, thereby providing valuable information for apple breeding.

Anthocyanin Biosynthesis and Methylation of the  Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple.

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 .

Adventitious root formation is essential for plant propagation, development, and response to various stresses. Reactive oxygen species (ROS) are essential for adventitious root formation. However, information on Respiratory Burst Oxidase Homolog (RBOH), a key enzyme that catalyzes the production ROS, remains limited in woody plants. Here, a total of 44 RBOH genes were identified from six Rosaceae species (Malus domestica, Prunus avium, Prunus dulcis 'Texas', Rubus occidentalis, Fragaria vesca and Rosa chinensis), including ten from M. domestica. Their phylogenetic relationships, conserved motifs and gene structures were analyzed. Exogenous treatment with the RBOH protein inhibitor diphenyleneiodonium (DPI) completely inhibited adventitious root formation, whereas exogenous H2O2 treatment enhanced adventitious root formation. In addition, we found that ROS accumulated during adventitious root primordium inducing process. The expression levels of MdRBOH-H, MdRBOH-J, MdRBOH-A, MdRBOH-E1 and MdRBOH-K increased more than two-fold at days 3 or 9 after auxin treatment. In addition, cis-acting element analysis revealed that the MdRBOH-E1 promoter contained an auxin-responsive element and the MdRBOH-K promoter contained a meristem expression element. Based on the combined results from exogenous DPI and H2O2 treatment, spatiotemporal expression profiling, and cis-element analysis, MdRBOH-E1 and MdRBOH-K appear to be candidates for the control of adventitious rooting in apple.

Genome-wide identification and characterization of Respiratory Burst Oxidase Homolog genes in six Rosaceae species and an analysis of their effects on adventitious rooting in apple.

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.

Deciphering the mechanism of malate accumulation in apple (Malus domestica Borkh.) fruits can help to improve their flavor quality and enhance their benefits for human health. Here, we analyzed malate content as a quantitative trait that is determined mainly by genetic effects. In a previous study, we identified an R2R3-MYB transcription factor named MdMYB44 that was a candidate gene in qtl08.1 (quantitative trait locus mapped to chromosome 8) of fruit malate content. In the present study, we established that MdMYB44 negatively regulates fruit malate accumulation by repressing the promoter activity of the malate-associated genes Ma1 (Al-Activated Malate Transporter 9), Ma10 (P-type ATPase 10), MdVHA-A3 (V-type ATPase A3), and MdVHA-D2 (V-type ATPase D2). Two single-nucleotide polymorphisms (SNPs) in the MdMYB44 promoter, SNP A/G and SNP T/-, were experimentally shown to associate with fruit malate content. The TATA-box in the MdMYB44 promoter in the presence of SNP A enhances the basal activity of the MdMYB44 promoter. The binding of a basic-helix-loop-helix transcription factor MdbHLH49 to the MdMYB44 promoter was enhanced by the presence of SNP T, leading to increased MdMYB44 transcript levels and reduced malate accumulation. Furthermore, MdbHLH49 interacts with MdMYB44 and enhances MdMYB44 activity. The two SNPs could be used in combination to select for sour or non-sour apples, providing a valuable tool for the selection of fruit acidity by the apple breeding industry. This research is important for understanding the complex molecular mechanisms of fruit malate accumulation and accelerating the development of germplasm innovation in apple species and cultivars.

Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.).

Color is an important trait for horticultural crops. Carotenoids are one of the main pigments for coloration and have important implications for photosynthesis in plants and benefits for human health. Here, we identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factor named MdAP2-34 in apple (Malus domestica Borkh.). MdAP2-34 expression exhibited a close correlation with carotenoid content in 'Benin Shogun' and 'Yanfu 3' fruit flesh. MdAP2-34 promotes carotenoid accumulation in MdAP2-34-OVX transgenic apple calli and fruits by participating in the carotenoid biosynthesis pathway. The major carotenoid contents of phytoene and β-carotene were much higher in overexpressing MdAP2-34 transgenic calli and fruit skin, yet the predominant compound of lutein showed no obvious difference, indicating that MdAP2-34 regulates phytoene and β-carotene accumulation but not lutein. MdPSY2-1 (phytoene synthase 2) is a major gene in the carotenoid biosynthesis pathway in apple fruit, and the MdPSY2-1 gene is directly bound and transcriptionally activated by MdAP2-34. In addition, overexpressing MdPSY2-1 in apple calli mainly increases phytoene and total carotenoid contents. Our findings will advance and extend our understanding of the complex molecular mechanisms of carotenoid biosynthesis in apple, and this research is valuable for accelerating the apple breeding process.

An apple (Malus domestica) AP2/ERF transcription factor modulates carotenoid accumulation.

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
Rapid and Inexpensive Method of Loading Fluorescent Dye into Pollen Tubes and Root Hairs. PloS one\| PubMed ID: 27055240	2016
The role of Ca and Ca channels in the gametophytic self-incompatibility of Pyrus pyrifolia. Cell calcium\| PubMed ID: 27397621	2016
The changes of intracellular calcium concentration and distribution in the hard end pear (Pyrus pyrifolia cv. 'Whangkeumbae') fruit. Cell calcium\| PubMed ID: 29604960	2018
Soil amendment alters soil physicochemical properties and bacterial community structure of a replanted apple orchard. Microbiological research\| PubMed ID: 30269849	2018
Genome-wide identification and transcript analysis of TCP transcription factors in grapevine. BMC genomics\| PubMed ID: 31664916	2019
RNA-Seq analysis of compatible and incompatible styles of Pyrus species at the beginning of pollination. Plant molecular biology\| PubMed ID: 31872308	2020
Genome-wide identification and analysis of B-BOX gene family in grapevine reveal its potential functions in berry development. BMC plant biology\| PubMed ID: 32054455	2020
Anthocyanin Biosynthesis and Methylation of the Promoter Are Associated with the Red Blushed-Skin Mutant in the Red Striped-Skin "Changfu 2" Apple. Journal of agricultural and food chemistry\| PubMed ID: 32207980	2020
Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of . Horticulture research\| PubMed ID: 32528703	2020
Genome-wide identification and characterization of Respiratory Burst Oxidase Homolog genes in six Rosaceae species and an analysis of their effects on adventitious rooting in apple. PloS one\| PubMed ID: 32976536	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
Genetic variation in the promoter of an R2R3-MYB transcription factor determines fruit malate content in apple (Malus domestica Borkh.). Plant physiology\| PubMed ID: 33624810	2021
An apple (Malus domestica) AP2/ERF transcription factor modulates carotenoid accumulation. Horticulture research\| PubMed ID: 34611138	2021
Accumulation of Abnormal Amyloplasts in Pulp Cells Induces Bitter Pit in . Frontiers in plant science\| PubMed ID: 34630490	2021

Yongzhang Wang

Biography

JoVE Journal Publications

Other Publications