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

ABSTRACT: 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.

SUBMITTER: Qiu L

PROVIDER: S-EPMC7261807 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundApple replant disease is a soilborne disease caused by Fusarium proliferatum f. sp. malus domestica strain MR5 (abbreviated hereafter as Fpmd MR5) in China. This pathogen causes root tissue rot and wilting leaves in apple seedlings, leading to plant death. A comparative transcriptome analysis was conducted using the Illumina Novaseq platform to identify the molecular defense mechanisms of the susceptible M.26 and the resistant M9T337 apple rootstocks to Fpmd MR5 infection.ResultsApproximately 518.1 million high-quality reads were generated using RNA sequencing (RNA-seq). Comparative analysis between the mock-inoculated and Fpmd MR5 infected apple rootstocks revealed 28,196 significantly differentially expressed genes (DEGs), including 14,572 up-regulated and 13,624 down-regulated genes. Among them, the transcriptomes in the roots of the susceptible genotype M.26 were reflected by overrepresented DEGs. MapMan analysis indicated that a large number of DEGs were involved in the response of apple plants to Fpmd MR5 stress. The important functional groups identified via gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were responsible for fundamental biological regulation, secondary metabolism, plant-pathogen recognition, and plant hormone signal transduction (ethylene and jasmonate). Furthermore, the expression of 33 up-regulated candidate genes (12 related to WRKY DNA-binding proteins, one encoding endochitinase, two encoding beta-glucosidases, ten related to pathogenesis-related proteins, and eight encoding ethylene-responsive transcription factors) were validated by quantitative real-time PCR.ConclusionRNA-seq profiling was performed for the first time to analyze response of apple root to Fpmd MR5 infection. We found that the production of antimicrobial compounds and antioxidants enhanced plant resistance to pathogens, and pathogenesis-related protein (PR10 homologs, chitinase, and beta-glucosidase) may play unique roles in the defense response. These results provide new insights into the mechanisms of the apple root response to Fpmd MR5 infection.

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

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