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

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<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>
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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

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 &#39;Fuyudori&#39; and &#39;228&#39; 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&#183;L-1 &#177; 0.05 mg&#183;L-1 to 29.18 mg&#183;L-1 &#177; 0.43 mg&#183;L-1 after 5 min of oxygen bubbling. Following the isolation process, the yield of protoplasts was 1.04 x 107 for &#39;Fuyudori&#39; and 4.00 x 106 protoplasts&#183;g&#8722;1 fresh weight for &#39;228&#39; 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="Figure 1" class="xfigimg" src="/files/ftp_upload/67444/67444fig01.jpg" /> 
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.remotexs.ntu.edu.sg/files/ftp_upload/67444/67444fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67444/67444fig02v2.jpg" /> 
Figure 2: Protoplast isolation from leaf tissue of cabbage. (A) The appearance of commercial cabbage varieties &#39;Fuyudori&#39; and &#39;228&#39; 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 &#39;Fuyudori&#39; and &#39;228&#39; cabbage varieties and were observed under a microscope. Scale bar: 200 &#181;m. (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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67444/67444fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67444/67444fig03.jpg" /> 
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 &#39;Fuyudori&#39; cabbage protoplasts. (B) A total of 5 &#181;g of reporter plasmids are transfected into &#39;Fuyudori&#39; 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&#183;mL-1), OxyFluor (0.6 units&#183;mL-1), and sodium sulfite (1 g&#183;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 &#34;CK&#34; group represents the control group without any hypoxia treatment. After treatment, protoplasts are harvested for a dual luciferase assay. Each value represents the mean &#177; SD, with n = 4. Different letters indicate significant differences (P &#60; 0.05) based on a one-way ANOVA with LSD post-hoc test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/67444/Table 1.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.remotexs.ntu.edu.sg/files/ftp_upload/67444/Plasmids.zip" target="_blank">Please click here to download this File.</a>

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

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

Nanyang Technological University

Research

JoVE Journal

Biology

426 Views.  National Taiwan University. We onderzoeken hoe kool op moleculair niveau reageert op een laag zuurstofgehalte. Omdat kool moeilijk te verkrijgen is, ontwikkelen we een systeem met geïsoleerde koolcellen. Door deze reservecellen, ook wel protoplasten genoemd, bloot te stellen aan zuurstofarme omstandigheden, kunnen we hun stressrespons gemakkelijk onderzoeken.Werken met protoplast dat in oplossing is gesuspendeerd om zuurstofarme reacties te bestuderen, is lastig. De grote uitdag...

Video: Ontwikkeling van een koolprotoplastsysteem voor het bestuderen van hypoxietolerantie in brassica

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Developmental Biology

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

Environment

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

A Strategy to Validate the Role of Callose-mediated Plasmodesmal Gating in the Tropic Response

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The establishment of an auxin gradient is a key event leading to phototropism and gravitropism. Previously, polar auxin transport (PAT) was shown to establish an auxin gradient in different cellular domains of plants. However, Han et al. recently demonstrated that for proper auxin gradient formation, plasmodesmal callose-mediated symplasmic connectivity between the adjacent cells is also a critical factor. In this manuscript, the strategy to elucidate the role of particular genes, which can affect phototropism and gravitropism by altering the symplasmic connectivity through modulating plasmodesmal callose synthesis, is discussed. The first step is to screen aberrant tropic responses from 3-day-old etiolated seedlings of mutants or over-expression lines of a gene along with the wild type. This preliminary screening can lead to the identification of a range of genes functioning in PAT or controlling symplasmic connectivity. The second screening involves the sorting of candidates that show altered tropic responses by affecting symplasmic connectivity. To address such candidates, the movement of a symplasmic tracer and the deposition of plasmodesmal callose were examined. This strategy would be useful to explore new candidate genes that can regulate symplasmic connectivity directly or indirectly during tropic responses and other developmental processes.

This article describes the methods for screening the genes controlling plasmodesmal permeability and hence auxin gradient during tropic response. This includes the measurement of the degree of tropic response in hypocotyl of Arabidopsis thaliana and checking plasmodesmal permeability by 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) loading and finally callose level assessment.

The plant hormone auxin plays an important role in many growth and developmental processes, including tropic responses to light and gravity. The ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several crops, including lettuce and tomato. Within the plant research community, numerous hydroponic systems have been designed to study plant responses to biotic and abiotic stresses. Here we present a hydroponic protocol that can be easily implemented in laboratories interested in pursuing studies on plant mineral nutrition.
This protocol describes the hydroponic system set up in detail and the preparation of plant material for successful experiments. Most of the materials described in this protocol can be found outside scientific supply companies, making the set up for hydroponic experiments less expensive and convenient.
The use of a hydroponic growth system is most advantageous in situations where the nutrient media need to be well controlled and when intact roots need to be harvested for downstream applications. We also demonstrate how nutrient concentrations can be modified to induce plant responses to both essential nutrients and toxic non-essential elements.

Here, we present an easy-to-follow protocol to establish a successful hydroponic system for plant nutrition studies. This protocol has been extensively tested in Arabidopsis and can easily be adapted to other plant species to study specific nutritional requirements or the effect of non-essential elements on plant growth and development.

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.

Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to Plasmodiophora brassicae

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls ...

Evaluation of Photosynthetic Efficiency in Photorespiratory Mutants by Chlorophyll Fluorescence Analysis

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the enzymes and genes important for photosynthesis and photorespiration have been well studied for decades, but some aspects of these biochemical pathways and their crosstalk with several subcellular processes are not yet fully understood. Much of the work that has identified the genes and proteins important in plant metabolism has been conducted under highly controlled environments that may not best represent how photosynthesis and photorespiration function under natural and farming environments. Considering that abiotic stress results in impaired photosynthetic efficiency, the development of a high-throughput screen that can monitor both abiotic stress and its impact on photosynthesis is necessary.
Therefore, we have developed a relatively fast method to screen for abiotic stress-induced changes to photosynthetic efficiency that can identify uncharacterized genes with roles in photorespiration using chlorophyll fluorescence analysis and low CO2 screening. This paper describes a method to study changes in photosynthetic efficiency in transferred DNA (T-DNA) knockout mutants in Arabidopsis thaliana. The same method can be used for screening ethyl methanesulfonate (EMS)-induced mutants or suppressor screening. Utilizing this method can identify gene candidates for further study in plant primary metabolism and abiotic stress responses. Data from this method can provide insight into gene function that may not be recognized until exposure to increased stress environments.

We describe an approach to measure changes in photosynthetic efficiency in plants after treatment with low CO2 using chlorophyll fluorescence.

Photosynthesis and photorespiration represent the largest carbon fluxes in plant primary metabolism and are necessary for plant survival. Many of the ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

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

Samenvatting

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