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

Summary

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.

Abstract

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.

Introduction

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 losses^1,2. 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 plants^4,5 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 imaging^14,15. 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.

Protocol

Two commercial cabbage (B. oleracea var. capitata) cultivars were utilized in this study: 'Fuyudori' and '228'. 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

1. Sow 'Fuyudori' and '228' 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.
2. Grow cabbage seedlings in a growth room at 22 °C with a 16/8 h light-dark cycle. Maintain the light intensity of 100 µmol m^-2·s^-1 (white fluorescent tube) during the light phase.
3. 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, 'Fuyudori' seeds need to be sown approximately 2 days earlier than '228' seeds due to their different growth rates. This adjustment ensures uniform growth and maturity among the cabbage plants at the desired stage.

2. Cabbage protoplasts isolation

1. Prepare 12.5 mL of enzyme solution (Table 1) before each protoplast isolation.
   1. Pre-heat a solution with 10 mM MES (pH 5.7) and 0.6 M of mannitol at 55 °C. Continue warming the solution at 55 °C for 10 min after adding 1.5% Cellulase R10 and 0.75% Macerozyme R10.
   2. After the enzyme solution cools down to room temperature (25 °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 µm 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.
2. 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.
3. After vacuum infiltration in the dark for 30 min (Figure 2B), 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, 'Fuyudori' requires a longer digestion time (16 h) compared to '228' (4 h) due to thicker leaves of 'Fuyudori'.
4. 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.
5. Release the protoplast suspension by gentle swirling or using an orbital shaker. Filter the cell suspension through a 70 µm cell strainer into a 50 mL conical tube.
   NOTE: The isolated protoplasts are capable of passing through a 70 µm 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.
6. Centrifuge the protoplasts solution with 150 x g for 2 min at 4 °C then carefully remove the supernatant. 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·s^-1. Centrifuge the tube at 150 x g for 2 min at 4 °C, and repeat this washing procedure again to ensure complete removal of the enzyme solution.
7. 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.
8. 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).
9. Measure the protoplast concentration with a hemocytometer and adjust the final concentration to 4 x 10⁵ protoplasts·mL^-1 using MMG solution.

3. Protoplasts transfection

1. Mix a total volume of 10 µL of plasmid (5-10 µg) (Supplementary File 1) with 100 µL 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'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 µg of plasmid exhibit a proven transfection efficiency ranging from 30% to 40%.
2. Add an equal volume (110 µL) 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.
3. Incubate the protoplast mixture at room temperature in the dark for 10 min, then add 440 µL of W5 solution to terminate the reaction.
4. Centrifuge the transfected protoplasts at 150 x g for 2 min at 4 °C and resuspend the pellet in 750 µL of oxygen-enriched W5 solution. Transfer the resuspended protoplasts to a 6-well tissue culture plate pre-coated with 1% BSA. 
   NOTE: W5 solution used for cell incubation must be bubbled with oxygen using an oxygen concentrator (~90% oxygen) connected to an air stone for 5 min (Figure 2D). Following this oxygenation process, the final dissolved oxygen concentration in the W5 solution increased from 7.84 mg·L-¹ ± 0.05 mg·L^-1 to 29.18 mg·L^-1 ± 0.43 mg·L^-1, as measured with a dissolved 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.

4. Hypoxia treatment on cabbage protoplasts

1. 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.
   1. For chemically induced hypoxia on cabbage protoplasts, add OxyFluor, EC-Oxyrase, and sodium sulfite to the W5 protoplast solution.
      NOTE: Using 0.6 units·mL^-1 EC-oxyrase, 0.6 units·mL^-1 OxyFluor, and 1 g·mL^-1 sodium sulfite in W5 were the tested conditions capable of inducing BoADH1 and BoSUS1L promoter (Figure 3A) with low damage to protoplast integrity in the two examined cabbage varieties.
   2. For oxygen-consuming bag-induced hypoxia, place two oxygen absorber packs in a 3.5 L anaerobic jar to create a hypoxic environment.
2. Harvest hypoxia-treated protoplasts by centrifuging at 150 x g for 2 min at 4 °C. Freeze the collected protoplasts using liquid nitrogen and store them in a -80 °C freezer for subsequent assays.

5. Dual luciferase assay

1. Perform the dual-luciferase reporter assay according to the manufacturer's instructions with minor modification.
   1. Resuspend cabbage protoplasts in 50 µL of 1x passive lysis buffer and vortex for 10 s.
   2. Incubate disrupted protoplasts on ice for 10 min and collect the supernatant after centrifugation at 10,000 x g for 10 min at 4 °C.
   3. Add 20 µL of cell lysate to each well of the microplate. Dispense 100 µL of Luciferase assay reagent II into the wells and measure firefly luciferase activity using a microplate reader.
   4. Add 100 µL of the commercially available assay reagent to each well and measure Renilla luciferase activity using a microplate reader.

Results

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 experience...

Discussion

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·g⁻¹·FW of mesophyll tissue from both cabbage cultivars (Figure 2E,F). This ...

Materials

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