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....

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 co.......

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