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. 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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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The authors declare that they have no conflicts of interest with the contents of this article.

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 ...

As a important mineral element, calcium plays a key role in the growth and the quality control of tree fruits such as apple, it can regulate the hardness, sugar content, storage period, and the physiological disorder during storage. Calcium imaging is the most western method to detect dynamic changes in calcium ions in the cytoplasm. Small molecule fluorescent indicators, such as fluo-4/AM have calcium ion-specific selectivity and can be noninvasively loaded by esterification incubation, which is flexible, rapid, and non-cytotoxic.This protocol will introduce a method for loading small molecule chemical fluorescent reagent fluo-4/AM into plant protoplasts. Protoplast Extraction Add 0.5 milliliter of enzyme solution into a 1.5 milliliter centrifuge tube. Pick a healthy and ripe apple, then slice the pulp into 10 times 5 times 1 cubic milliliter size.Place the pulp pieces into enzymatic solution, then close the tube. Incubate tube at 28 degrees for one hour. Shacked at 70 rpm in dark.After that, wash the pulp pieces, aspirate all the enzymatic solution and then add 0.5 milliliter basic solution. Centrifuge at a speed of 300 g for 2 minutes. Protoplasts suspension can be obtained by aspirating the bottom solution.Small-molecule chemical fluorescence reagent staining. Prepare the fluo-4/AM loading solution with a 2 millimolar fluo-4/AM, 20%ethyl127, and the 10X phosphate-buffered saline in a one to one to two ratio. Add 1 microliter loading solution of fluo-4/AM to 99 microliter protoplast suspension.The final concentration of fluorescent dyes is 5 micromolar. Mix the solution then close the tube. At the same time, lanthanum iron the calcium or uniqueness of the small molecular chemical fluorescent agent.Add one microliter or 10 microliter more lanthanum iron and then one microliter loading solution of fluo-4/AM to 98 microliter protoplasts suspension. The final concentration of lanthanum iron is 100 micromolar. Mix the solution then close the tube.Incubate at 37 degrees for 30 minutes in dark. After that, centrifuge at the speed of 300 G for two minutes. Aspirate to 17 microliters solution and add a 17 microliter basic solution.Incubate the protoplast suspension at 37 degrees for 30 minutes to completely After that, aspirate 50 microliter protoplasts suspension and drip on to a slide. Observe under fluorescence microscope. Select the 20x the magnification and the GFP channel to observe.Uniformly adjust the brightness to 0.5. A critical step in this proposed method is to wash the stain in the protoplasts and incubate them for 30 minutes. Insufficient washing causes excessive backer on the fluorescence during observation and incubation allows for better loading or the probe into the cytoplasm.Protoplasts Viability Assay Take 99 microliter protoplasts suspension after incubating at 37 degrees for 30 minutes. Add 1 ml at the FD solution to protoplasts suspension. Mix the solution by pipetting up and down several times and then close the tube.Stay at room temperature for five minutes in dark. Prepare the slides and observe them on the fluorescence microscope with GFP channel. We present to you results Following figure one.We use the enzymatic method all obtaining protoplasts from the pulp. Some protoplasts had a vacuole while others did not. The protoplasts were stained with FD for five minutes and show the florescent in cytoplasm indicating that high temperature, 37 degrees does not affect the viability or protoplasts Figure two.The protoplasts exhibited no fluorescence in one of the calcium irons fluorescence indicator or it's not loaded into them. Than in fluo-4/AM was loaded into the protoplast, the cytoplast, but not a vacuole became fluorescent. Lanthanum Irons which effectively block calcium channels are the cell membrane were added to the Protoplast fluo-4/AM loading.At the final concentration of 100 micromolar lanthanum irons significantly reduce the calcium fluorescence intensity. This results further demonstrated that fluo-4/AM effectively stay in the calcium irons in the cytoplast and it show dynamic changes in calcium irons content. Figure three The fluo-4/AM results were analyzed using IMH approved plus 6.0 software fluo-4/AM staining with addition of lanthanum irons significantly reduced the florescence value of cytoplasmic calcium irons.In this video, were all protoplasts were obtained by enzymatic hydrolysis. We successfully loaded the fluorescent probes into a protoplasts at 37 degrees. More over, the method did not affect the viability of protoplasts.In summary, the method described here in kind detailed. The dynamic changes in cytoplasmic calcium ions in apple pulp cells thereby providing 10 equal supports for the further purpose of calcium related studies.

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Research

JoVE Journal

Biology

4.0K 조회수.  Qingdao Agricultural University. 중요한 미네랄 요소로서 칼슘은 사과와 같은 나무 과일의 성장과 품질 관리에 중요한 역할을 하며, 저장 시 경도, 당도, 저장 기간 및 생리장애를 조절할 수 있다. 칼슘 이미징은 세포질에서 칼슘 이온의 동적 변화를 감지하는 가장 서쪽 방법입니다. 형광4/AM과 같은 소분자 형광 지표는 칼슘 이온 특이적 선택성을 가지며 유연하고 신속한 비세포 독성인 에스테르화 인큐베이?...