Name: Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica (Video) | JoVE Journal
Uploaded: 2024-09-20T15:00:00.000+00:00
Duration: 8 min 9 s
Description: Learn how to develop a cabbage protoplast system for studying hypoxia tolerance in Brassica using enzymatic digestion and reporter assays. This method enables analysis of flooding stress responses at the molecular level.

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5 minutes

This work was supported by the National Science and Technology Council (MOST 111-2313-B-002-029- and NSTC 112-2313-B-002-050-MY3). For Figure 1, the experimental icons were sourced from BioRender.com.

Two commercial cabbage (B. oleracea var. capitata) cultivars were utilized in this study: &#39;Fuyudori&#39; and &#39;228&#39;. A graphical representation of the protocol workflow is shown in Figure 1. The details of the reagents and the equipment used in this study are listed in the Table of Materials.
1. Preparation of cabbage seedlings
<ol>
	<li>Sow &#39;Fuyudori&#39; and &#39;228&#39; cabbage seeds using commercial plant substrate into round square-shaped holes (D 4.5 cm x H 4.5 cm) of 48-well plug trays (L 49 cm x W 28 cm x H 5 cm). Embed the seeds approximately 1 cm deep into the growing medium.</li>
	<li>Grow cabbage seedlings in a growth room at 22 &#176;C with a 16/8 h light-dark cycle. Maintain the light intensity of 100 &#181;mol m-2&#183;s-1 (white fluorescent tube) during the light phase.</li>
	<li>After 2-3 weeks of growth, use cabbage seedlings at the 2-leaf stage for further mesophyll protoplast isolation. 
	NOTE: The suggested size of cabbage seedlings is a 2-leaf stage, in which the second true leaf is fully expanded, but the third true leaf is not expanded, and its length is shorter than 1 cm (Figure 2A). To synchronize the developmental stages of the cabbage seedlings, &#39;Fuyudori&#39; seeds need to be sown approximately 2 days earlier than &#39;228&#39; seeds due to their different growth rates. This adjustment ensures uniform growth and maturity among the cabbage plants at the desired stage.</li>
</ol>
2. Cabbage protoplasts isolation
<ol>
	<li>Prepare 12.5 mL of enzyme solution (Table 1) before each protoplast isolation.
	<ol>
		<li>Pre-heat a solution with 10 mM MES (pH 5.7) and 0.6 M of mannitol at 55 &#176;C. Continue warming the solution at 55 &#176;C for 10 min after adding 1.5% Cellulase R10 and 0.75% Macerozyme R10.</li>
		<li>After the enzyme solution cools down to room temperature (25 &#176;C), add 10 mM of CaCl2 and 0.1% bovine serum albumin (BSA). Filter sterilize the prepared enzyme solution into a 9 cm Petri dish using a 0.22 &#181;m syringe filter. 
		NOTE: Pre-warming of solution increases enzyme solubility and inactivates protease. The digestion effects vary depending on the different types of cellulase enzymes. The combination of Cellulase R10 and Macerozyme R10 is suitable for cabbage protoplast isolation.</li>
	</ol>
	</li>
	<li>Collect the second newly expanded true leaves from five to eight cabbage seedlings, and slice them into 0.5-1.0 mm strips using a sharp razor blade. Immediately transfer the leaf strips into the freshly prepared enzyme solution. 
	NOTE: Selecting healthy and designated leaves from cabbage at the proper stage is crucial. The second newly expanded true leaves from cabbage seedlings at the 2-leaf stage provide the highest protoplast isolation efficiency. Over-mature plants, cotyledons, or aged leaves are not recommended for protoplast isolation. Five to twelve well-expanded true leaves can successfully yield cabbage protoplasts in 12.5 mL of enzyme solution. The number of leaves required for protoplast isolation depends on the desired quantity of protoplasts needed. Typically, protoplasts obtained from five to eight leaves are sufficient for subsequent experimental procedures.</li>
	<li>After vacuum infiltration in the dark for 30 min (Figure 2B),&#160;keep the cabbage leaf strips immersed in the enzyme solution (Figure 2C) in the dark at room temperature for a further 4-16 h. 
	NOTE: Enzyme digestion time may vary among cabbage cultivars and should be optimized according to the specific genotype. For instance, &#39;Fuyudori&#39; requires a longer digestion time (16 h) compared to &#39;228&#39; (4 h) due to thicker leaves of &#39;Fuyudori&#39;.</li>
	<li>Dilute the protoplasts-containing solution with an equal volume of W5 solution, which consists of 2 mM of MES (pH 5.7), 154 mM of NaCl, 125 mM of CaCl2, 5 mM of KCl, and 5 mM of glucose (see Table 1 for preparation), to stop enzymatic digestion.</li>
	<li>Release the protoplast suspension by gentle swirling or using an orbital shaker. Filter the cell suspension through a 70 &#181;m cell strainer into a 50 mL conical tube. 
	NOTE: The isolated protoplasts are capable of passing through a 70 &#181;m cell strainer, while undigested plant debris is retained by the strainer and subsequently removed from the suspension. Cell strainers can be reused and kept in 75% ethanol after washing, but it is necessary to fully rinse cell strainers with W5 solution to remove residual ethanol before use.</li>
	<li>Centrifuge the protoplasts solution with 150 x g for 2 min at 4 &#176;C then carefully remove the supernatant.&#160;Gently wash the pelleted protoplasts by adding 10 mL of W5 solution along the wall of the conical tube at an approximate flow rate of 1 mL&#183;s-1. Centrifuge the tube at 150 x g for 2 min at 4 &#176;C, and repeat this washing procedure again to ensure complete removal of the enzyme solution.</li>
	<li>Resuspend the protoplasts in the W5 solution and place them on ice for 30 min. After the ice incubation, remove the supernatant by a pipette 1 mL at a time until all the supernatant is removed.</li>
	<li>Resuspend the protoplasts in an ice-pre-chilled MMG solution composed of 4 mM of MES (pH 5.7), 0.4 M of mannitol, and 15 mM of MgCl2 (see Table 1 for preparation).</li>
	<li>Measure the protoplast concentration with a hemocytometer and adjust the final concentration to 4 x 105 protoplasts&#183;mL-1 using MMG solution.</li>
</ol>
3. Protoplasts transfection
<ol>
	<li>Mix a total volume of 10 &#181;L of plasmid (5-10 &#181;g) (Supplementary File 1) with 100 &#181;L of protoplasts (4 x 104 protoplasts) and place on ice for 10 min. 
	NOTE: For transfection-grade plasmid purification, a commercially available plasmid kit is used following the manufacturer&#39;s instructions. Approximately 4 x 104 protoplasts are typically employed in a dual-luciferase reporter assay. For larger-scale experiments, a higher quantity of protoplasts is available for transfection. Specifically, around 2 x 105 protoplasts transfected with 10 &#181;g of plasmid exhibit a proven transfection efficiency ranging from 30% to 40%.</li>
	<li>Add an equal volume (110 &#181;L) of freshly prepared PEG solution (see Table 1 for preparation) containing 40% Polyethylene Glycol 4000 (PEG4000), 0.1 M of CaCl2, and 0.2 M of mannitol into the protoplast solution and mix gently.</li>
	<li>Incubate the protoplast mixture at room temperature in the dark for 10 min, then add 440 &#181;L of W5 solution to terminate the reaction.</li>
	<li>Centrifuge the transfected protoplasts at 150 x g for 2 min at 4 &#176;C and resuspend the pellet in 750 &#181;L of oxygen-enriched W5 solution. Transfer the resuspended protoplasts to a 6-well tissue culture plate pre-coated with 1% BSA.&#160; 
	NOTE: W5 solution used for cell incubation must be&#160;bubbled with oxygen using an oxygen concentrator&#160;(~90% oxygen) connected to an air stone for 5 min (Figure 2D). Following this oxygenation process, the final&#160;dissolved oxygen concentration in the W5 solution increased from 7.84 mg&#183;L-1 &#177; 0.05 mg&#183;L-1 to 29.18&#160;mg&#183;L-1 &#177; 0.43 mg&#183;L-1, as measured with a dissolved&#160;oxygen meter. The volume of W5 solution for resuspension of transfected protoplast depends on the type of tissue cell culture plate used. For 12-well or 24-well cell culture plates, it is advisable to reduce the volume of W5 solution to mitigate hypoxia stress during incubation due to the deeper depth within the wells. For BSA coating, each well of the culture plate received 1 mL of 1% BSA solution, which was allowed to coat the wells for 10 s. The BSA solution was then discarded from each well, and the wells were subsequently refilled with W5 solution. This procedure aimed to create a temporary non-adherent surface using BSA, effectively preventing protoplasts from sticking to the plastic surface of the culture plate during subsequent experimental steps.</li>
</ol>
4. Hypoxia treatment on cabbage protoplasts
<ol>
	<li>Conduct chemical or physically induced hypoxia treatments immediately after protoplast transfection. 
	NOTE: It is recommended that hypoxia treatment be applied immediately after protoplast transfection. Prolonged incubation of protoplasts may diminish the subsequent hypoxia response.
	<ol>
		<li>For chemically induced hypoxia on cabbage protoplasts, add OxyFluor, EC-Oxyrase, and sodium sulfite to the W5 protoplast solution. 
		NOTE: Using 0.6 units&#183;mL-1 EC-oxyrase, 0.6 units&#183;mL-1 OxyFluor, and 1 g&#183;mL-1 sodium sulfite in W5 were the tested conditions capable of inducing BoADH1 and BoSUS1L promoter&#160;(Figure 3A) with low damage to protoplast integrity in the two examined cabbage varieties.</li>
		<li>For oxygen-consuming bag-induced hypoxia, place two oxygen absorber packs in a 3.5 L anaerobic jar to create a hypoxic environment.</li>
	</ol>
	</li>
	<li>Harvest hypoxia-treated protoplasts by centrifuging at 150 x g for 2 min at 4 &#176;C. Freeze the collected protoplasts using liquid nitrogen and store them in a -80 &#176;C freezer for subsequent assays.</li>
</ol>
5. Dual luciferase assay
<ol>
	<li>Perform the dual-luciferase reporter assay according to the manufacturer&#39;s instructions with minor modification.
	<ol>
		<li>Resuspend cabbage protoplasts in 50 &#181;L of 1x passive lysis buffer and vortex for 10 s.</li>
		<li>Incubate disrupted protoplasts on ice for 10 min and collect the supernatant after centrifugation at 10,000 x g for 10 min at 4 &#176;C.</li>
		<li>Add 20 &#181;L of cell lysate to each well of the microplate. Dispense 100 &#181;L of Luciferase assay reagent II into the wells and measure firefly luciferase activity using a microplate reader.</li>
		<li>Add 100 &#181;L of the commercially available assay reagent to each well and measure Renilla luciferase activity using a microplate reader.</li>
	</ol>
	</li>
</ol>

Efficient Cabbage Protoplast System for Studying Flooding Tolerance Mechanisms

<ol><li>Tanoue, M., Hirabayashi, Y., Ikeuchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global-scale+river+flood+in+the+last+50+years%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Global-scale river flood in the last 50 years.</a> Sci Rep. 6 (1), 36021(2016).</li>
<li>Voesenek, L. aC. J., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flood+adaptive+traits+and+processes%3A+An+overview%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">Flood adaptive traits and processes: An overview.</a> New Phytol. 206 (1), 57-73 (2015).</li>
<li>Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+transduction+in+maize+and+Arabidopsis+mesophyll+protoplasts%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Signal transduction in maize and Arabidopsis mesophyll protoplasts.</a> Plant Physiol. 127 (4), 1466-1475 (2001).</li>
<li>Chen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+highly+efficient+transient+protoplast+system+for+analyzing+defense+gene+expression+and+protein-protein+interactions+in+rice%5BTitle%5D)+AND+%22Mol+Plant+Pathol%22%5BJournal%5D)">A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice.</a> Mol Plant Pathol. 7 (5), 417-427 (2006).</li>
<li>Yoo, S. -D., Cho, Y. -H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+mesophyll+protoplasts%3A+A+versatile+cell+system+for+transient+gene+expression+analysis%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2 (7), 1565-1572 (2007).</li>
<li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+protoplast+isolation%2C+transformation%2C+and+its+application+in+sugarcane+%28Saccharum+spontaneum+L%29%5BTitle%5D)+AND+%22Crop+J%22%5BJournal%5D)">Optimization of protoplast isolation, transformation, and its application in sugarcane (Saccharum spontaneum L).</a> Crop J. 9 (1), 133-142 (2021).</li>
<li>Adedeji, O. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+protocol+for+efficient+protoplast+isolation+and+transient+gene+expression+in+carnation%5BTitle%5D)+AND+%22Sci+Hortic%22%5BJournal%5D)">Optimization of protocol for efficient protoplast isolation and transient gene expression in carnation.</a> Sci Hortic. 299, 111057(2022).</li>
<li>Lin, H. -Y., Chen, J. -C., Fang, S. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protoplast+transient+expression+system+to+enable+molecular%2C+cellular%2C+and+functional+studies+in+Phalaenopsis+orchids%5BTitle%5D)+AND+%22Front+Plant+Sci%22%5BJournal%5D)">A protoplast transient expression system to enable molecular, cellular, and functional studies in Phalaenopsis orchids.</a> Front Plant Sci. 9, (2018).</li>
<li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+highly+efficient+mesophyll+protoplast+isolation+and+PEG-mediated+transient+expression+system+in+eggplant%5BTitle%5D)+AND+%22Sci+Hortic%22%5BJournal%5D)">A highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in eggplant.</a> Sci Hortic. 304, 111303(2022).</li>
<li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+protoplast+isolation+system+for+functional+gene+expression+and+characterization+using+petals+of+Camellia+oleifera%5BTitle%5D)+AND+%22Plant+Physiol+Biochem%22%5BJournal%5D)">Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia oleifera.</a> Plant Physiol Biochem. 201, 107885(2023).</li>
<li>Guo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+efficient+isolation+of+Populus+mesophyll+protoplasts+and+its+application+in+transient+expression+assays%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.</a> PLOS One. 7 (9), e44908(2012).</li>
<li>Gottschald, O. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TIAR+and+TIA-1+mRNA-binding+proteins+co-aggregate+under+conditions+of+rapid+oxygen+decline+and+extreme+hypoxia+and+suppress+the+HIF-1%CE%B1+pathway%5BTitle%5D)+AND+%22J+Mol+Cell+Biol%22%5BJournal%5D)">TIAR and TIA-1 mRNA-binding proteins co-aggregate under conditions of rapid oxygen decline and extreme hypoxia and suppress the HIF-1α pathway.</a> J Mol Cell Biol. 2 (6), 345-356 (2010).</li>
<li>Cho, H. -Y., Chou, M. -Y., Ho, H. -Y., Chen, W. -C., Shih, M. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethylene+modulates+translation+dynamics+in+Arabidopsis+under+submergence+via+GCN2+and+EIN2%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2.</a> Sci Adv. 8 (22), eadm7863(2022).</li>
<li>Boudreau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excitation+light+dose+engineering+to+reduce+photo-bleaching+and+photo-toxicity%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Excitation light dose engineering to reduce photo-bleaching and photo-toxicity.</a> Sci Rep. 6 (1), 30892(2016).</li>
<li>Shcherbakova, D. M., Cox Cammer, N., Huisman, T. M., Verkhusha, V. V., Hodgson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+multiplex+imaging+and+optogenetics+of+Rho+GTPases+enabled+by+near-infrared+FRET%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.</a> Nat Chem Biol. 14 (6), 591-600 (2018).</li>
<li>Jiang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sodium+sulfite+is+a+potential+hypoxia+inducer+that+mimics+hypoxic+stress+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22J+Biol+Inorg+Chem%22%5BJournal%5D)">Sodium sulfite is a potential hypoxia inducer that mimics hypoxic stress in Caenorhabditis elegans.</a> J Biol Inorg Chem. 16 (2), 267-274 (2011).</li>
<li>Lin, C. -C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SUB1A-1+anchors+a+regulatory+cascade+for+epigenetic+and+transcriptional+controls+of+submergence+tolerance+in+rice%5BTitle%5D)+AND+%22PNAS+Nexus%22%5BJournal%5D)">SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional controls of submergence tolerance in rice.</a> PNAS Nexus. 2 (7), pgad229(2023).</li>
<li>Zhang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bis-molybdopterin+guanine+dinucleotide+modulates+hemolysin+expression+under+anaerobiosis+and+contributes+to+fitness+in+vivo+in+uropathogenic+Escherichia+coli%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">Bis-molybdopterin guanine dinucleotide modulates hemolysin expression under anaerobiosis and contributes to fitness in vivo in uropathogenic Escherichia coli.</a> Mol Microbiol. 116 (4), 1216-1231 (2021).</li>
<li>Liu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+light+and+hypoxia-induced+gene+zmPORB1+determines+tocopherol+content+in+the+maize+kernel%5BTitle%5D)+AND+%22Sci+China+Life+Sci%22%5BJournal%5D)">The light and hypoxia-induced gene zmPORB1 determines tocopherol content in the maize kernel.</a> Sci China Life Sci. 67 (3), 435-448 (2024).</li>
</ol>

The authors declare no competing interest.

This protocol presents a streamlined method for protoplast isolation from two commercial cabbage cultivars. The efficacy of this method is primarily assessed through two critical quality control parameters: the yield of viable protoplasts and the efficiency of protoplast transfection. Implementing this protocol resulted in a yield exceeding 4.00 x 106 protoplasts&#183;g&#8722;1&#183;FW of mesophyll tissue from both cabbage cultivars (Figure 2E,F). This yield is comparable to those reported in other species, including eggplant, carnation, sugarcane, and Camellia oleifera, where protoplast yields ranged from 2.5 x 106 to 1.4 x 107 protoplasts&#183;g&#8722;1&#183;FW6,7,9,17. These results suggest the protocol&#39;s potential applicability across diverse genetic backgrounds with B. oleracea.
The protocol&#39;s success hinges on several critical factors. Foremost is the utilization of specified leaves from healthy cabbage seedlings at an appropriate growth stage (Figure 2A). Using older or unhealthy plants may result in poor-quality protoplasts. It is also crucial to carefully cut the leaf tissue into 0.5-1.0 mm strips and avoid crushing it with a blunt blade. The enzyme digestion process, lasting between 4-16 h, is another critical step. Digestion periods shorter than 4 h may lead to incomplete digestion, while those exceeding 16 h of digestion risk cellular stress. Notably, genotypes with thicker leaves, such as &#39;Fuyudori&#39;, may benefit from extended digestion times to maximize protoplast yield. In contrast, &#39;228&#39; required only 4 h for complete digestion. In addition, a wide range of digestion times can provide greater flexibility in experimental scheduling. For instance, a 4 h digestion allows for protoplast isolation and subsequent transient assays to be completed within a single day. Conversely, a 16 h digestion allows for&#160;the initiation of enzyme digestion in the evening and enables the completion of subsequent steps on the following day, which can enhance convenience when applying this protocol. Moreover, it is crucial to handle the protoplasts gently throughout the entire protoplast isolation process due to their fragility and susceptibility to mechanical stress.
To simulate anaerobic conditions representative of flooding stress, this protocol utilized common oxygen-scavenging agents and oxygen-consuming bags to create hypoxic environments. To enhance the system&#39;s effectiveness, an oxygen machine is employed to oxygenate the W5 solution (Figure 2D), aiming to reduce background hypoxic response signals in cabbage protoplasts during their pre-treatment incubation period. A 5 min oxygenation process significantly elevates the dissolved oxygen level from 7.84 mg&#183;L-1 &#177; 0.05 mg&#183;L-1 to 29.18 mg&#183;L-1 &#177; 0.43 mg&#183;L-1, indicating a pronounced effect in alleviating hypoxic conditions. Among the oxygen-scavenging methods used in this study, the oxygen absorber packs exhibited superior performance, inducing the highest promoter activity in two anaerobic response core genes (Figure 3). Furthermore, this method offers the additional advantage of scalability, allowing simultaneous treatment of multiple samples through the use of oxygen-consuming bags in a single anaerobic jar, as opposed to the individual application of oxygen-scavenging agents to each cell culture plate. In summary, the oxygen absorber packs&#160;provide a convenient and effective platform suitable for evaluating the response of cabbage protoplasts under hypoxic conditions.
While this protocol demonstrates several advantages, it is important to acknowledge its limitations, particularly the relatively modest transfection efficiency of cabbage protoplasts. Although this efficiency does not reach the 90% reported for Arabidopsis5, it nonetheless still proves sufficient for successful subsequent studies on promoter activity (Figure 3), indicating significant potential for functional analysis in this system.
Given the global importance of cabbage as a major vegetable crop, the integration of this protocol with hypoxia treatments and genetic analysis techniques holds promise to gain further insights into regulatory mechanisms in cabbage under flooding conditions. Such insights could potentially accelerate the development of flood-tolerant cabbage varieties, addressing a critical need in the face of extreme weather and increasing flood events.

Global climate change has exacerbated flooding, which has emerged as an increasingly critical issue worldwide. Recent decades have witnessed an upward trend in the frequency of flooding events, resulting in substantial crop losses1,2. Cabbage (Brassica oleracea var. capitata L.), a vegetable of significant global importance, is susceptible to the adverse effects of heavy rainfall, necessitating the development of flooding-tolerant cabbage cultivars to ensure sustainable production in the face of extreme weather events. Therefore, understanding the molecular mechanisms associated with flooding stress in cabbage is essential to meet this challenge.
To understand gene regulation mechanisms in plants under submerged conditions, transgenic lines are widely used for genetic functional studies. However, this approach is constrained by high costs, time-intensive transformation, and subculture processes, as well as low transformation efficiencies in many crop species, necessitating the development of alternative methodologies. Protoplast-based transient expression systems have been widely applied in plant molecular research as a versatile and efficient alternative. These systems facilitate investigations into promoter activity, signaling pathways in response to environmental cues, protein-protein or protein-DNA interactions, and subcellular localization3. The establishment of protoplast transient expression systems has been reported not only in model plants4,5 but also in economically important crops such as sugarcane6, carnation7, Phalaenopsis orchids8, and eggplant9. Moreover, these systems have been successfully implemented in woody plants, including Camellia oleifera10and Populus trichocarpa11. However, protocols for applying protoplast systems to study leafy vegetables under submergence-induced hypoxia stress are limited. Therefore, an integrated protocol has been developed in this work for those interested in studying hypoxia responses in leafy vegetables using a cabbage protoplast transient expression system.
To carry out the submergence-induced hypoxia response at the cellular level, several oxygen-scavenging methodologies have been employed in previous studies to simulate hypoxic environments. These include the use of EC-Oxyrase, OxyFluor, sodium sulfite, and oxygen-consuming bags. EC-Oxyrase is typically used for anaerobic treatment in human cell lines12 and Arabidopsis protoplasts13. OxyFluor has been found to be effective in mitigating photobleaching caused by reactive oxygen species during live-cell fluorescence imaging14,15. Sodium sulfite has been employed in the anaerobic treatment of nematodes16 and, more recently, in rice protoplasts, in conjunction with techniques such as chromatin immunoprecipitation (ChIP) assays17. Oxygen absorber packs, primarily used for anaerobic bacterial culture18, have also demonstrated efficacy in inducing the activation of ZmPORB1 promoters in maize protoplasts under anaerobic conditions19.
The present work aims to establish a robust pipeline for cabbage protoplast isolation and transient expression. Subsequently, the efficacy of various oxygen-adjusting treatments was evaluated by assessing the promoter activity of anaerobic response genes using dual-luciferase assays. The protocol developed in this study is anticipated to be valuable for future research related to submergence or hypoxia stress in Brassica systems.

<table><tbody><tr><td>2-(N-morpholino) ethanesulfonic Acid (MES)</td><td>PhytoTech Labs</td><td>M825</td><td>For enzyme solution preparation</td></tr><tr><td>228 cabbage seeds</td><td>Takii &amp;amp; Co., Ltd. (Kyoto, Japan)</td><td></td><td></td></tr><tr><td>50 mL Conical Tube</td><td>SPL Life Sciences</td><td>50050</td><td>For enzyme solution preparation</td></tr><tr><td>6-well tissue culture plate</td><td>Alpha Plus</td><td>16106</td><td>For protoplast incubation</td></tr><tr><td>70 &amp;mu;m cell strainer</td><td>Sorfa&amp;nbsp;</td><td>SCS701</td><td>For protoplast filtration</td></tr><tr><td>9-cm Petri dish</td><td>Alpha Plus</td><td>16001</td><td>For enzymatic digestion</td></tr><tr><td>Anaerobic jar</td><td>HIMEDIA</td><td>Anaerobic Jar 3.5 L</td><td>For hypoxia treatment&amp;nbsp;</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td>For W5 solution preparation and culture plate coating</td></tr><tr><td>Calcium chloride</td><td>J.T.Baker</td><td>131301</td><td>For W5 solution and PEG solution preparation</td></tr><tr><td>Cellulase R10</td><td>Yakult</td><td></td><td>For enzyme solution preparation</td></tr><tr><td>Desiccator</td><td>Tarsons&amp;nbsp;</td><td>402030</td><td>For vacuum infiltration</td></tr><tr><td>D-Glucose</td><td>Bioshop</td><td>GLU501</td><td>For W5 solution preparation</td></tr><tr><td>Dissolved oxygen meter</td><td>Thermo Scientific</td><td>Orion Star A223</td><td>For oxygen measurement</td></tr><tr><td>D-Mannitol</td><td>Sigma-Aldrich</td><td>M1902</td><td>For enzyme solution, PEG solution, and MMG solution preparation</td></tr><tr><td>Dual-Luciferase Reporter Assay System</td><td>Promega</td><td>E1960</td><td>For Dual-luciferase reporter assay</td></tr><tr><td>EC-Oxyrase</td><td>Oxyrase Inc.</td><td>EC-0005</td><td>For hypoxia treatment</td></tr><tr><td>Fuyudori cabbage seeds</td><td>Kobayashi Seed Co., Ltd. (Kakogawashi, Japan)</td><td></td><td></td></tr><tr><td>High-Speed refrigerated centrifuge</td><td>Hitachi</td><td>CR21GIII</td><td>For protoplast harvest</td></tr><tr><td>Macerozyme R10&amp;nbsp;</td><td>Yakult</td><td></td><td>For enzyme solution preparation</td></tr><tr><td>Magnesium chloride</td><td>Alfa Aesar</td><td>12315</td><td>For MMG solution preparation</td></tr><tr><td>Microcentrifuge</td><td>Hitachi</td><td>CT15RE</td><td>For protoplast harvest</td></tr><tr><td>Microplate</td><td>Greiner</td><td>655075</td><td>For Dual-luciferase reporter assay</td></tr><tr><td>Microplate Reader</td><td>Molecular Devices</td><td>SpectraMax Mini</td><td>For Dual-luciferase reporter assay</td></tr><tr><td>Millex 0.22 &amp;mu;m syringe filter</td><td>Merck</td><td>SLGP033RS</td><td>For enzyme solution preparation</td></tr><tr><td>Oil Free Vacuum Pump</td><td>Rocker&amp;nbsp;</td><td>Rocker 300</td><td>For vacuum infiltration</td></tr><tr><td>OxyFluor</td><td>Oxyrase Inc.</td><td>OF-0005</td><td>For hypoxia treatment</td></tr><tr><td>Oxygen absorber pack</td><td>Mitsubishi Gas Chemical Company</td><td>AnaeroPack, MGCC1</td><td>For hypoxia treatment</td></tr><tr><td>Oxygen concentrator</td><td>UTMOST PERFECT</td><td>AII-X</td><td>For oxygen-bubbling in W5 solution</td></tr><tr><td>Plant substrate</td><td>Klasmann-Deilmann</td><td>Potgrond H substrate</td><td>For cabbage seedlings preparation</td></tr><tr><td>Plasmid Midi Kit</td><td>QIAGEN</td><td>12145</td><td>For purification of transfection-grade plasmid DNA&amp;nbsp;</td></tr><tr><td>Polyethylene Glycol 4000</td><td>Fluka</td><td>81240</td><td>For protoplast transfection</td></tr><tr><td>Potassium chloride</td><td>J.T.Baker</td><td>304001</td><td>For W5 solution preparation</td></tr><tr><td>Razor blade</td><td>Gillette</td><td></td><td>For cabbage leaf strips preparation</td></tr><tr><td>Sodium chloride</td><td>Bioshop</td><td>SOD002</td><td>For W5 solution preparation</td></tr><tr><td>Sodium sulfite</td><td>Sigma-Aldrich</td><td>S0505</td><td>For hypoxia treatment</td></tr><tr><td>Water Bath</td><td>Yihder</td><td>BU-240D</td><td>For enzyme solution preparation</td></tr></tbody></table>

development of a cabbage protoplast system for studying hypoxia tolerance in brassica

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia stress. To identify mechanisms of flooding tolerance in cabbages, a versatile platform for genetic functional studies is needed to overcome the transformation-recalcitrant nature of cabbages. In this study, a cabbage protoplast transient expression system and a corresponding protoplast hypoxia induction protocol were developed. This protocol achieved a high yield and integrity of protoplast isolation from cabbage leaves, with a transfection efficiency exceeding 40% using optimized enzymatic conditions. To alleviate potential hypoxic influence before treatments, the W5 solution was bubbled with oxygen gas to increase dissolved oxygen levels. Several chemicals for adjusting oxygen levels and physiological oxygen-scavenging treatments were tested, including EC-Oxyrase, OxyFluor, sodium sulfite, and an oxygen absorber pack. Dual-luciferase assays showed that promoters of anaerobic respiration response genes BoADH1 and BoSUS1L were activated in cabbage protoplasts after hypoxia treatments, with the highest induction level observed after treatment with the oxygen absorber pack. In summary, the cabbage protoplast transient expression system combined with hypoxia treatment demonstrates an efficient and convenient platform. This platform can facilitate studies of gene function and molecular mechanisms associated with hypoxia responses in cabbages.

This work successfully developed a transient expression system utilizing cabbage protoplasts (see Figure 1 for workflow). Protoplasts were isolated from 2- to 3-week-old cabbage true leaves of appropriate size (Figure 2A) from commercial cabbage cultivars 'Fuyudori' and '228' using cellulase/macerozyme digestion and dark vacuum infiltration (Figure 2B,C). To mitigate the hypoxic conditions experienced by submerged protoplasts during incubation, the W5 solution was oxygenated using an air stone bubbler connected to an oxygen machine (Figure 2D). This process significantly elevated the dissolved oxygen concentration in the W5 solution from 7.84 mg·L-1 ± 0.05 mg·L-1 to 29.18 mg·L-1 ± 0.43 mg·L-1 after 5 min of oxygen bubbling. Following the isolation process, the yield of protoplasts was 1.04 x 107 for 'Fuyudori' and 4.00 x 106 protoplasts·g−1 fresh weight for '228' after an overnight digestion period (Figure 2E). Subsequently, GFP was used as a reporter gene to evaluate the transformation efficiency of the PEG-mediated transfection method, demonstrating a protoplast transfection efficiency in cabbage was above 40% in both cultivars (Figure 2F), thus confirming the viability of this system.
Furthermore, promoter constructs of cabbage anaerobic respiration response genes, BoADH1p::Luc and BoSUS1Lp::Luc vectors (Figure 3A), were introduced into cabbage protoplasts as reporters to evaluate the hypoxic induction effects of several treatments. Various oxygen-adjusting treatments were also applied to this transient expression model system. The results showed that all oxygen-scavenging treatments, with the exception of sodium sulfite, successfully induced a hypoxia response in cabbage protoplasts (Figure 3B). Specifically, the promoter activity of BoADH1 increased approximately 2.6-fold and 3.2-fold compared to the control following treatment with EC-Oxyrase and OxyFluor, respectively, and, notably, by more than 7.0-fold with oxygen absorber pack treatment (Figure 3B). Similarly, the BoSUS1L promoter activity rose about 1.7-fold and 1.8-fold with EC-Oxyrase and OxyFluor treatment, respectively, while oxygen absorber pack treatment induced a substantial increase of over 5.6-fold (Figure 3B). These results suggest that the oxygen absorber pack demonstrates superior efficacy in creating a hypoxic environment in this cabbage protoplast experimental system.
<img alt="Protoplast isolation diagram; cabbage leaves, enzyme solution, PEG-CaCl2 transfection process." class="xfigimg" src="/files/ftp_upload/67444/67444fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Graphical representation of the protoplast isolation workflow. This figure illustrates the step-by-step process involved in isolating protoplasts from cabbage leaf tissue. The workflow is visually depicted to provide a clear understanding of the procedure. <a href="https://www.jove.com/files/ftp_upload/67444/67444fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plant protoplast isolation process; vacuum infiltration, enzymatic digestion, oxygen bubbling setup." class="xfigimg" src="/files/ftp_upload/67444/67444fig02v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Protoplast isolation from leaf tissue of cabbage. (A) The appearance of commercial cabbage varieties 'Fuyudori' and '228' at the recommended growth stage is shown. The second newly fully expanded true leaves (indicated by the arrowhead) of cabbages in the 2-leaf stage are selected for protoplast isolation. Scale bar: 2 cm. (B) A vacuum pump and desiccator are employed for the vacuum infiltration process of cabbage leaf strips, with a 30 min infiltration performed before enzymatic digestion. (C) The condition of cabbage leaf strips during enzymatic digestion is depicted. Complete digestion of the leaf tissue requires a total of 4-16 h. Scale bar: 3 cm. (D) Photos demonstrate the use of an oxygen concentrator connected to an air stone to increase the dissolved oxygen concentration in the W5 solution. Scale bar: 5 cm. (E) Over 4.00 x 106 mesophyll protoplasts per gram of fresh weight (FW) were obtained from both 'Fuyudori' and '228' cabbage varieties and were observed under a microscope. Scale bar: 200 µm. (F) The transfection efficiency of protoplasts from both cabbage genotypes, using a constitutively expressed GFP reporter, is shown to be above 40%. Scale bar: 200 µm. <a href="https://www.jove.com/files/ftp_upload/67444/67444fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gene expression diagram with BoADH1, BoSUS1L promoters; bar graph results of luciferase activity." class="xfigimg" src="/files/ftp_upload/67444/67444fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Hypoxia response of cabbage protoplasts under oxygen-deficient conditions. (A) Hypoxia response gene constructs, BoADH1 and BoSUS1L promoters, are utilized as reporters to evaluate the hypoxia response in 'Fuyudori' cabbage protoplasts. (B) A total of 5 µg of reporter plasmids are transfected into 'Fuyudori' cabbage protoplasts, which are incubated in an oxygen-bubbled W5 solution before exposure to hypoxia treatments for 4 h. Oxygen levels are reduced during incubation by adding EC-oxyrase (0.6 units·mL-1), OxyFluor (0.6 units·mL-1), and sodium sulfite (1 g·mL-1) to the W5 solution. The oxygen absorber pack treatment is performed by placing two oxygen absorber bags in a 3.5 L anaerobic jar. The "CK" group represents the control group without any hypoxia treatment. After treatment, protoplasts are harvested for a dual luciferase assay. Each value represents the mean ± SD, with n = 4. Different letters indicate significant differences (P &lt; 0.05) based on a one-way ANOVA with LSD post-hoc test. <a href="https://www.jove.com/files/ftp_upload/67444/67444fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Preparation of enzyme solution, W5 solution, and MMG solution. <a href="https://www.jove.com/files/ftp_upload/67444/Table%201.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Plasmid details for protoplast transfection. <a href="https://www.jove.com/files/ftp_upload/67444/Plasmids.zip" target="_blank">Please click here to download this File.</a>

This protocol describes an efficient cabbage mesophyll protoplast system. Various oxygen-deficient treatments were tested, and the system showed a high activation of hypoxia-responsive genes, facilitating studies of the genetic and molecular mechanisms of flooding tolerance in Brassicaceae vegetables.

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia ...

development-cabbage-protoplast-system-for-studying-hypoxia-tolerance

Research

JoVE Journal

Biology

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Learn how to develop a cabbage protoplast system for studying hypoxia tolerance in Brassica using enzymatic digestion and reporter assays. This method enables analysis of flooding stress responses at the molecular level.

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

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