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 co...

<ol>
	<li>Tanoue, M., Hirabayashi, Y., Ikeuchi, H. Global-scale river flood&#160;vulnerability in the last 50 years. Sci Rep. 6 (1), 36021 (2016).</li>
	<li>Voesenek, L. a. C. J. Bailey-Serres, J. Flood adaptive traits and processes: An overview. New Phytol. 206 (1), 57-73 (2015).</li>
	<li>Sheen, J. Signal transduction in maize and Arabidopsis mesophyll protoplasts. Plant Physiol. 127 (4), 1466-1475 (2001).</li>
	<li>Chen, S. et al. A highly efficient transient protoplast system for analyzing defense gene expression and protein-protein interactions in rice. Mol Plant Pathol. 7 (5), 417-427 (2006).</li>
	<li>Yoo, S.-D., Cho, Y.-H., Sheen, J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat Protoc. 2 (7), 1565-1572 (2007).</li>
	<li>Wang, Q. et al. Optimization of protoplast isolation, transformation, and its application in sugarcane (Saccharum spontaneum L.). Crop J. 9 (1), 133-142 (2021).</li>
	<li>Adedeji, O. S. et al. Optimization of protocol for efficient protoplast isolation and transient gene expression in carnation. Sci Hortic. 299, 111057 (2022).</li>
	<li>Lin, H.-Y., Chen, J.-C., Fang, S.-C. A protoplast transient expression system to enable molecular, cellular, and functional studies in Phalaenopsis orchids. Front Plant Sci. 9,&#160;(2018).</li>
	<li>Wang, Y. et al. A highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in eggplant. Sci Hortic. 304, 111303 (2022).</li>
	<li>Lin, Z. et al. Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia oleifera. Plant Physiol Biochem. 201, 107885 (2023).</li>
	<li>Guo, J. et al. Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLOS One. 7 (9), e44908 (2012).</li>
	<li>Gottschald, O. R. et al. TIAR and TIA-1 mRNA-binding proteins co-aggregate under conditions of rapid oxygen decline and extreme hypoxia and suppress the HIF-1&#945; pathway. 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. Ethylene modulates translation dynamics in Arabidopsis under submergence via GCN2 and EIN2. Sci Adv. 8 (22), eabm7863 (2022).</li>
	<li>Boudreau, C. et al. Excitation light dose engineering to reduce photo-bleaching and photo-toxicity. Sci Rep. 6 (1), 30892 (2016).</li>
	<li>Shcherbakova, D. M., Cox Cammer, N., Huisman, T. M., Verkhusha, V. V., Hodgson, L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat Chem Biol. 14 (6), 591-600 (2018).</li>
	<li>Jiang, B. et al. Sodium sulfite is a potential hypoxia inducer that mimics hypoxic stress in Caenorhabditis elegans. J Biol Inorg Chem. 16 (2), 267-274 (2011).</li>
	<li>Lin, C.-C. et al. SUB1A-1 anchors a regulatory cascade for epigenetic and transcriptional controls of submergence tolerance in rice. PNAS Nexus. 2 (7), pgad229 (2023).</li>
	<li>Zhang, X. et al. Bis-molybdopterin guanine dinucleotide modulates hemolysin expression under anaerobiosis and contributes to fitness in vivo in uropathogenic Escherichia coli. Mol Microbiol. 116 (4), 1216-1231 (2021).</li>
	<li>Liu, N. et al. The light and hypoxia-induced gene zmPORB1 determines tocopherol content in the maize kernel. Sci China Life Sci. 67 (3), 435-448 (2024).</li>
</ol>

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 ...

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 oleifera10 and 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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 &#956;m cell strainer</td>
			<td>Sorfa&#160;</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&#160;</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&#160;</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&#160;</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 &#956;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&#160;</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&#160;</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 experience...

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.

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 ...