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

Published: November 6th, 2021



1College of Horticulture, Qingdao Agricultural University

Isolated protoplasts of apple pulp cells were loaded with a calcium fluorescent reagent to detect cytoplasmic Ca2+ concentration.

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 this study, we obtained viable protoplasts of pulp cells by enzymatic hydrolysis. Isolated protoplasts were incubated with the small-molecule fluorescent reagent (Fluo-4/AM) for 30 min at 37 °C. The fluorescent probes successfully stained cytosolic Ca2+ but did not accumulate in vacuoles. La3+, a Ca2+ channel blocker, decreased cytoplasmic fluorescence intensity. These results suggest that Fluo-4/AM can be used to detect changes in cytosolic Ca2+ in the fruit flesh. In summary, we present a method to effectively isolate protoplasts from flesh cells of the fruit and detect Ca2+ by loading a small-molecule calcium fluorescent reagent in the cytoplasm of pulp cells.

Ca2+ plays an important role in plant signal transduction and metabolism1,2. Further, it regulates fruit quality traits3,4, including hardness, sugar content, and susceptibility to physiological disorders during storage5,6. Cytoplasmic Ca2+ plays an important role in signal transduction and regulates plant growth and development7. Disturbance of cellular calcium homeostasis can induce bitter pit in apples8, brown spot disease....

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1. Protoplast extraction

  1. Prepare the basic solution: 20 mM CaCl2, 5 mM 2-(N-morpholino)ethanesulfonic acid, and 0.4 M D-sorbitol.
    NOTE: The pH of the basic solution was adjusted to 5.8 with 0.1 M Tris buffer, filtered through 0.22 µm water-soluble filters, and stored at 4 °C.
  2. Prepare the enzymatic solution: Mix 0.3%(w/v) Macerozyme R-10 and 0.5%(w/v) cellulase R-10 with the basic solution.
  3. Add 0.5 mL of enzymatic solution into a 1.5 mL centrifug.......

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Following the protocol described above, we used the enzymatic method to obtain viable protoplasts from the pulp (Figure 1). Some protoplasts had vacuoles, while others did not. While the protoplasts exhibited no fluorescence when the Ca2+ fluorescent indicator was not loaded into them. When Fluo-4/AM was loaded into the protoplasts, the cytoplasm, but not the vacuole, became fluorescent (Figure 2). This result indicated that Fluo-4/AM successfully sta.......

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In this study, viable protoplasts were obtained by enzymatic hydrolysis. Note that this method requires fresh apples. The present protocol allows for the rapid isolation of a large number of protoplasts from fruit pulp for use in research studies. The applicability of this method is not limited to 'Fuji'; the protoplasts of the apple pulp of 'Dounan' and 'Honey Crisp' can also be extracted through the same protocol (Supplementary Figure S4). The protoplast solution after enzymolys.......

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This work was supported by the Agricultural Variety Improvement Project of Shandong Province (2019LZGC007) and Fruit tree innovation team of Shandong modern agricultural industry technology system (SDAIT-06-05).


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Name Company Catalog Number Comments
10× phosphate-buffered saline Solarbio P1022 PBS (phosphate buffered solution) is a phosphate buffer solution, which can provide a relatively stable ionic environment and pH buffering capacity. It is a buffer salt solution often used in biology for molecular cloning and cell culture. The pH is 7.4. 
2-(N-morpholino)ethanesulfonic acid Solarbio M8010 Biological buffer
CaCl2·2H2O Solarbio C8370 Calcium chloride dihydrate is a white or gray chemical, mostly in granular form.
Cellulase R-10 Yakult Honsha MX7352 Degrade plant cell walls.
D-sorbitol Solarbio S8090 It has good moisturizing properties, prevents drying, and prevents sugar, salt, etc. from crystallizing.
F-127 Thermo Fisher Scientific P6867 Pluronic F-127 is a non-ionic, surfactant polyol (molecular weight of approximately 12500 Daltons), which has been found to be beneficial to promote the dissolution of water-insoluble dyes and other materials in physiological media. 
FDA Thermo Fisher Scientific F1303 FDA is a cell-permeant esterase substrate that can serve as a viability probe that measures both enzymatic activity, which is require to activate its fluorescence, and cell-membrane integrity, which is required for intracellular retention of their fluorescent product. 
Fluo-4/AM Thermo Fisher Scientific F14201 The green fluorescent calcium indicator Fluo-4/AM is an improved version of the calcium indicator Fluo-3/AM. The Fluo-4/AM loads faster and is brighter at the same concentration. It can be well excited with a 488 nm argon ion laser.
Fluorescence microscope Thermo Fisher EVOS Auto 2 Observe the fluorescence image.
Macerozyme R-10 Yakult Honsha MX7351 Degrade plant tissue to separate single cells.
Tris Solarbio T8060 It is widely used in the preparation of buffers in biochemistry and molecular biology experiments.

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