Name: staining the cytoplasmic ca2+ with fluo-4/am in apple pulp
Uploaded: 2021-11-06T13:00:00
Description: Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

This&#160;work was supported by the Agricultural Variety Improvement Project of Shandong Province (2019LZGC007)&#160;and Fruit tree innovation team of Shandong modern agricultural industry&#160;technology system (SDAIT-06-05).

1. Protoplast extraction
<ol>
	<li>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 &#181;m water-soluble filters, and stored at 4 &#176;C.</li>
	<li>Prepare the enzymatic solution: Mix 0.3%(w/v) Macerozyme R-10 and 0.5%(w/v) cellulase R-10 with the basic solution.</li>
	<li>Add 0.5 mL of enzymatic solution into a 1.5 mL centrifug...

<ol>
	<li>Hocking, B., Tyerman, S. D., Burton, R. A., Gilliham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruit+calcium:+Transport+and+physiology.">Fruit calcium: Transport and physiology.</a> Frontiers in Plant Science. 7, 569 (2016).</li><li>Li, J., Yang, H. -. q., Yan, T. -. l., Shu, H. -. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+indole+butyric+acid+on+the+transportation+of+stored+calcium+in+Malus+hupehensis+rhed.+Seedling.">Effect of indole butyric acid on the transportation of stored calcium in Malus hupehensis rhed. Seedling.</a> Agricultural Sciences in China. 5 (11), 834-838 (2006).</li><li>Gao, Q., Xiong, T., Li, X., Chen, W., Zhu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+and+calcium+sensors+in+fruit+development+and+ripening.">Calcium and calcium sensors in fruit development and ripening.</a> Scientia Horticulturae. 253, 412-421 (2019).</li><li>Barrett, D. M., Beaulieu, J. C., Shewfelt, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color,+flavor,+texture,+and+nutritional+quality+of+fresh-cut+fruits+and+vegetables:+desirable+levels,+instrumental+and+sensory+measurement,+and+the+effects+of+processing.">Color, flavor, texture, and nutritional quality of fresh-cut fruits and vegetables: desirable levels, instrumental and sensory measurement, and the effects of processing.</a> Critical Reviews in Food Science and Nutrition. 50 (5), 369-389 (2010).</li><li>Deell, J. R., Khanizadeh, S., Saad, F., Ferree, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+apple+fruit+firmness--a+review.">Factors affecting apple fruit firmness--a review.</a> Journal- American Pomological Society. 55 (1), 8-27 (2001).</li><li>Johnston, J., Hewett, E., Hertog, M. A. T. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+on+blossom-end+rot+incidence+in+two+tomato+cultivars+in+relation+to+calcium+nutrition+and+fruit+growth.">Comparative analysis on blossom-end rot incidence in two tomato cultivars in relation to calcium nutrition and fruit growth.</a> The Horticulture Journal. 87 (1), 97-105 (2018).</li><li>Yamane, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foliar+calcium+applications+for+controlling+fruit+disorders+and+storage+life+in+deciduous+fruit+trees.">Foliar calcium applications for controlling fruit disorders and storage life in deciduous fruit trees.</a> Japan Agricultural Research Quarterly. 48 (1), 29-33 (2014).</li><li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+in+neurons.">Imaging calcium in neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Bootman, M. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnesium+availability+regulates+the+development+of+root+hairs+in+Arabidopsis+thaliana+(L.)+Heynh.">Magnesium availability regulates the development of root hairs in Arabidopsis thaliana (L.) Heynh.</a> Plant Cell and Environment. 37 (12), 2795-2813 (2014).</li><li>Qiu, L., Wang, Y., Qu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading+calcium+fluorescent+probes+into+protoplasts+to+detect+calcium+in+the+flesh+tissue+cells+of+Malus+domestica.">Loading calcium fluorescent probes into protoplasts to detect calcium in the flesh tissue cells of Malus domestica.</a> Horticulture Research. 7, 91 (2020).</li><li>Boyer, J., Liu, R. 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D., Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+intracellular+calcium.">Measurement of intracellular calcium.</a> Physiological Reviews. 79 (4), 1089-1125 (1999).</li><li>Qu, H., Shang, Z., Zhang, S., Liu, L., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+hyperpolarization-activated+calcium+channels+in+apical+pollen+tubes+of+Pyrus+pyrifolia.">Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.</a> New Phytologist. 174 (3), 524-536 (2007).</li><li>Hadjantonakis, A. K., Pisano, E., Papaioannou, V. 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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 &#39;Fuji&#39;; the protoplasts of the apple pulp of &#39;Dounan&#39; and &#39;Honey Crisp&#39; can also be extracted through the same protocol (Supplementary Figure S4). The protoplast solution after enzymolys...

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 in pears9, and umbilical rot in tomatoes10, affecting fruit quality and causing severe economic losses3,11. Calcium imaging has sufficient spatial and temporal resolution and is an important method for observing Ca2+ dynamics in living cells12,13.
At present, there are two main methods for intracellular calcium imaging in live cells: one employs chemical small-molecular fluorescent probes14, and the other is the gene encoding sensor (GECI)15,16. Given the difficulty of establishing a stable transgenic system in fruit trees and longer fruit development, GECIS is unsuitable for fruit Ca2+ fluorescence imaging.
Small-molecule fluorescent probes such as Fluo-4/AM have a particular advantage: their AM ester form (cell-permeable acetoxymethyl ester derivative) can be readily bulk-loaded into living cells without the need for transfection, which makes it flexible, rapid, and non-cytotoxic17. Fluo-4/AM could successfully be loaded into the pollen tube of Pyrus pyrifolia18 and Petunia,19 as well as into guard cells20 and root hair of Arabidopsis21.
At present, there are few reports on the calcium fluorescence staining of pulp cells22. As an important mineral element, calcium plays a key role in the growth and quality control of tree fruits such as apples. Apple trees are globally recognized as an important economic species, and apples are considered a healthy food23. In this study, we obtained viable protoplasts from apple fruit pulp through enzymatic hydrolysis and then loaded small-molecule fluorescent reagents into the cytoplasm to detect Ca2+.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10&#215; phosphate-buffered saline</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td>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.&#160;</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)ethanesulfonic acid</td>
			<td>Solarbio</td>
			<td>M8010</td>
			<td>Biological buffer</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Solarbio</td>
			<td>C8370</td>
			<td>Calcium chloride dihydrate is a white or gray chemical, mostly in granular form.</td>
		</tr>
		<tr>
			<td>Cellulase R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7352</td>
			<td>Degrade plant cell walls.</td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Solarbio</td>
			<td>S8090</td>
			<td>It has good moisturizing properties, prevents drying, and prevents sugar, salt, etc. from crystallizing.</td>
		</tr>
		<tr>
			<td>F-127</td>
			<td>Thermo Fisher Scientific</td>
			<td>P6867</td>
			<td>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.&#160;</td>
		</tr>
		<tr>
			<td>FDA</td>
			<td>Thermo Fisher Scientific</td>
			<td>F1303</td>
			<td>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.&#160;</td>
		</tr>
		<tr>
			<td>Fluo-4/AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Thermo Fisher</td>
			<td>EVOS Auto 2</td>
			<td>Observe the fluorescence image.</td>
		</tr>
		<tr>
			<td>Macerozyme R-10</td>
			<td>Yakult Honsha</td>
			<td>MX7351</td>
			<td>Degrade plant tissue to separate single cells.</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td>It is widely used in the preparation of buffers in biochemistry and molecular biology experiments.</td>
		</tr>
	</tbody>
</table>

staining the cytoplasmic ca2+ 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 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 &#176;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.

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...

Watch this Scientific Journal Video about Staining the Cytoplasmic Ca2+ with Fluo-4/AM in Apple Pulp at JoVE.com

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

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

Staining the Cytoplasmic Ca2+ 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 ...

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Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...