Plant materials and growth conditions
Soybean (Glycine max (L.) Merr. ‘Enrei’) seeds used in this study were collected from field-grown plants. The mutant line EnT-6693 was isolated by amplicon sequencing from an EMS-treated mutant population as previously described27 using primer pair LUS-Gln729stop-053 (Supplementary Table 7). We identified EnT-6693(WT) and EnT-6693 (lus1) from the progeny of EnT-6693 because the mutation of GmLUS1 in EnT-6693 was a heterozygote. Genome editing of GmLUS1 and GmLUS2 in soybean (G. max (L.) Merr. ‘Williams 82’) using the CRISPR/Cas9 system was performed by BIOGLE GeneTech (http://www.biogle.cn/, Hangzhou, China). Target sequences are shown in Extended Data Fig. 9; T1 seeds were purchased from BIOGLE GeneTech. Before the start of the experiments, we extracted DNA from each individual plant and checked the genotype. PCR was performed using PrimeSTAR®ฎ MAX DNA polymerase (Takara Bio, Shiga, Japan) and specific primers are listed in Supplementary Table 7. Sequencing of amplified DNA was performed by Eurofins Genomics (https://eurofinsgenomics.jp/jp/home.aspx, Tokyo, Japan).
Soybean seedlings were grown following a previously established method13. Plastic pots (300 mL) were filled with 200 mL silica sand (18–26 mesh). A 2 cm deep hole was dug in each pot, into which a single soybean seed was sown and covered with a small amount of the silica sand. Seedlings were grown at 25°C under 14 h light/10 h dark conditions for approximately 10 d (light conditions [photosynthetically active radiation, PAR]: 180–200 µmol/m2/s1) until unifoliate leaves were fully expanded (approximately 10 days old). The soil was waterlogged by raising the deionized water (DI) levels, which were maintained at 3 cm above the soil surface for 0 d, 1 d, 3 d, 5 d, 7 d, 9 d, and 14 d. Hypocotyls from 0.5–1.5 cm above the soil surface were harvested for further analysis.
For radial oxygen loss (ROL) measurements, seeds were sterilized using CruiserMaxx (Syngenta Japan, Tokyo, Japan) and imbibed in vermiculite by wetting with water for 3 days. Seedlings were then transferred to an aerated 25% nutrient solution and grown hydroponically with aeration for 4 days (14 h light, 28 ℃/10 h dark, 17 ℃). The nutrient solution at full concentration consisted of 0.5 mM KH2PO4, 3.0 mM KNO3, 4.0 mM Ca(NO3)2 mM, 1.0 MgSO4 mM, 37.5 µM FeNa3EDTA, 23.0 µM H3BO3, 4.5 µM MnCl2, 4.0 µM ZnSO4, 1.5 µM CuSO4, and 0.05 µM MoO3. The pH of the solution was buffered with 2.5 mM MES (2-(N-morpholino) ethanesulfonic acid) adjusted with KOH to a pH of 6.3. One week after germination, the seedlings were transplanted into pots containing 5 L of stagnant deoxygenated nutrient solution, which contained the same full-strength nutrient solution as described above and 0.1% (w/v) agar; this solution was deoxygenated by bubbling with nitrogen gas34. The seedlings were grown under a stagnant deoxygenated nutrient solution for 14 days, and the solution was replenished every 7 days.
Methylene blue staining
After the unifoliate leaves were fully expanded, the seedlings were grown under drained and waterlogged soil conditions for 7 days. The seedlings were dug out from the soil and immersed in methylene blue solution containing 13 mg/L methylene blue (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), 0.1% agar, and 135 mg/L sodium hydrosulfite (Fujifilm Wako Pure Chemical Corporation).
Preparation for LCM
Hypocotyl segments from 0.5–1.5 cm above the soil surface were fixed with 100% ethanol. Fixed segments were embedded in paraffin using an H2850 Microwave Tissue Processor (Energy Beam Sciences, East Granby, CT, USA) as previously described35; 14 µm-thick cross sections of the hypostyle were prepared and mounted on the PEN Membrane Frame Slides (Thermo Fisher Scientific, Waltham, MA, USA). Then, phellogen, AP, cortex, and stele were isolated by Veritas Laser Microdissection System LCC1704 (Molecular Devices, San Jose, CA, USA), as previously described35.
RNA extraction
Total RNA was extracted from each LM-isolated tissue using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific) and RNA quality was evaluated using the Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. RNA quantity was determined using the RiboGreen RNA Quantification Kit (Thermo Fisher Scientific) and an FP-6500 Spectrofluorometer (Jasco Inc., Tokyo, Japan).
Total RNA was extracted from hypocotyls using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
Microarray analysis
Total RNA samples (10 ng each) were labeled with a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer’s instructions. Aliquots of Cy5-labeled and Cy3-labeled cRNA (825 ng each) were used for hybridization in a 4 × 44k soybean Gene Expression Microarray (Agilent Technologies) that contains 42,748 oligo probes to soybean genes.
Six biological replicates were used, and the labels of Cy3 and Cy5 were interchanged in three of the six replicates of the experiment. Microarray signal intensities were digitized, and the log2 ratio and p-values were obtained by Feature Extraction software v.10.5.1.1 (Agilent Technologies). A complete set of microarray data was deposited in the Gene Expression Omnibus repository under accession no. GSE216072. We selected genes that showed FC ≥ 2.0 or ≤ 0.5 in expression between phellogen and cortex or AP and cortex, and p-values < 0.05 in all six replicates (Supplementary Tables 1–4). The FC of each probe was calculated using the average of six replicates. Phytozome IDs were obtained by the BLAST sequence of each probe. TAIR accession and description were obtained from Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html).
Extraction and determination of lupeol, betulin, and betulinic acid
Hypocotyl segments from 0.5–3 cm from the soil surface were cut from seedlings grown under drained or waterlogged soil conditions and immediately frozen with liquid nitrogen. Segments were freeze-dried using FreeZone 1 Liter Benchtop Freeze Dry System (Labconco, MO, USA). Freeze-dried segments were ground using Multi Beads Shocker (Yasui-Kikai, Osaka, Japan). Triterpenoids were extracted and analyzed following a previously described method with minor modifications36.
Quantitative real-time PCR (qRT-PCR) analysis
Relative mRNA levels were investigated with qRT-PCR using a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). First-strand cDNA was synthesized using Superscript III (Thermo Fisher Scientific) from 20 ng of total RNA extracted from LM-isolated tissues and 2 µg from hypocotyls. SYBR Premix Ex Taq II (Takara Bio) was used for subsequent PCR amplification with appropriate primers (Supplementary Table 7): initial denaturation (95 ℃ for 20 s), 40 cycles of denaturation (95 ℃ for 3 s), annealing, and extension (60 ℃ for 30 s).
In situ hybridization
Hypocotyl segments from 0.5–1.5 cm above the soil surface were fixed with fixative solution (4% paraformaldehyde, 2.5% glutaraldehyde, 50 mM Na-P buffer pH 7.2, 0.8 mM NaOH). Fixed segments were embedded in paraffin using an H2850 Microwave processor (Energy Beam Sciences) as previously described35. Sections (10 µm thickness) were cut with a rotary microtome. In situ hybridization was performed as described previously37. The coding region of GmLUS1 (Glyma.20G192700) was amplified from soybean cDNA by PCR using appropriate primers (Supplementary Table 7). PCR fragment was cloned to the pCR 4Blunt-TOPO vector (Thermo Fisher Scientific). To produce digoxigenin-labeled GmLUS1 sense and antisense probes, a 2,263 bp DNA fragment was amplified by PCR from a cDNA clone and used as a template for in vitro transcription with a Maxi Script in vitro transcription kit (Thermo Fisher Scientific). Hybridization was conducted at 50°C overnight.
LUS activity assay in GmLUS1 and GmLUS2
Coding regions of GmLUS1 and GmLUS2 (Glyma.08G027000) were amplified from soybean cDNA by PCR using appropriate primers (Supplementary Table 7). The PCR products were cloned into the pENTR/D-TOPO vector using pENTR Directional TOPO Cloning Kits (Thermo Fisher Scientific) following the manufacturer’s instructions. To obtain the expression vectors, pYES3-ADH-GW was used for the Gateway LR reactions by LR Clonase II Enzyme Mix (Thermo Fisher Scientific). We performed yeast in vivo assay as described previously with minor modifications31,38. pYES3-ADH-GmLUS1 and pYES3-ADH-GmLUS2 were transformed to Saccharomyces cerevisiae INVSc1 (Thermo Fisher Scientific).
Yeast culture extracts (starting volume: 5 mL) were prepared using 3 mL ethyl acetate (Wako, Osaka, Japan), followed by 1 min of vortexing and 30 min of sonication (70% intensity; Sharp Co., Osaka, Japan). After centrifuging at 2,000 rpm for 5 min, the organic phase was transferred to a silica-gel column (6 cc; Waters Corp., Milford, MA, USA) and washed with 10 mL ethyl acetate. The samples were then placed into an evaporator for 60 min. After resuspending the obtained pellet in 300 µL chloroform–methanol, 100 µL of the mixture was transferred to a vial and placed in an evaporator for 30 min. Finally, the pellet was trimethylsilylated with 50 µL of N-methyl-N-(trimethylsilyl)trifluoro acetamide (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 80°C. The evaporated samples were stored at 4°C until needed.
GC-MS was performed using a JMS-AMSUN200 mass spectrometer (JEOL Ltd., Tokyo, Japan) connected to a gas chromatograph (6890A; Agilent Technologies) with a DB1-HT (30 m × 0.25 mm, 0.1 µm film thickness; J&W Scientific, Folsom, CA, USA) capillary column. The injection temperature was 250°C. The column temperature program was as follows: 80°C for 1 min, followed by an increase to 300°C at a rate of 20°C/min, and a hold at 300°C for 20 or 28 min. The carrier gas was He, and the flow rate was 1.2 or 1.0 mL/min, respectively; the interface temperature was 300°C with a spitless injection21. Peaks were identified by comparing the Rt and mass spectrum with that of the lupeol authentic standard.
Cryo-SEM
After 14 days of waterlogging treatment, hypocotyl segments from 1.0 − 1.5 cm above the soil surface were isolated using a razor blade. The segment was mounted on the metal stage with Tissue-Tek O.C.T. Compound (Sakura Finetek, Tokyo, Japan) and immediately frozen with liquid nitrogen. The surfaces of the AP cells were observed with a field emission scanning electron microscope (S-4300K, Hitachi, Tokyo, Japan) equipped with a cryo-stage (-150 to -120°C). The accelerating voltage was 3 kV.
X-ray CT
After 14 days of waterlogging treatment, 0.4 cm hypocotyl segments were isolated using a razor blade. The segment was inserted into the 5 cm plastic straw (φ10 mm), and both ends were closed with wet absorbent cotton. This straw was longitudinally mounted on the stage by sealing putty, and scanned with a micro-focus X-ray CT (ScanXmate-L090T, ComscanTecno, Kanagawa, Japan). Micro-CT scans were taken at 35 keV and 200 µA; the resolution was 7 µm/pixel, and the rotation step was 0.15°. The total scan time was approximately 20 min, resulting in 1,200 cross-sectional image slices of 1,296 × 1,152 pixels each. Volume renderings and quantitative calculation of the intercellular space on the sample were performed by 3D image segmentation and isosurface representations with Image-Pro 3D (ver.10, Media Cybernetics, USA). The cuboid region (96 × 96 × 296 pixels), which was 30 pixels and 150 − 180 pixels far from the stele, was extracted as “Inside” and “Outside,” respectively (Extended Data Fig. 7).
Porosity measurement
After 14 days of waterlogging treatment, AP-well-developed hypocotyl segments from 0.5 − 1.5 cm above the soil surface were isolated using a razor blade. The porosity of hypocotyl segments was measured and calculated using the buoyancy method39,40.
TTC staining
After 14 days of waterlogging treatment, AP-well-developed hypocotyl segments from 0.5 − 1.5 cm from the soil surface were isolated using a razor blade. The segments were immersed in the TTC solution (0.06% TTC, 0.05% Tween 20, 100 mM Na-P buffer pH 7.0), vacuumed for 10 min, and incubated for 1 h at 42 ℃. After washing the segments using deionized water, 200 µm cross sections were prepared with a plant microtome (MTH-1, Nippon Medical & Chemical Instruments co., Osaka, Japan). Under stereo microscopy, the area of AP was isolated with a razor blade. For the extraction of pigment, isolated sections were incubated in the 200 µL 95%(v/v) ethanol. The reduction of TTC was expressed as the absorbance of the extracted solutions at 520 nm in a spectrophotometer (DU800, Beckman Coulter Inc., CA, USA).
Measurement of oxygen leakage from the adventitious roots
ROL measurement was performed using a root-sleeving oxygen electrode. Root-sleeving (i.e., cylindrical platinum) oxygen electrodes enable quantification of ROL at selected positions along roots in an oxygen-free medium41 Twenty-one-day-old plants were immersed until 2 cm below cotyledonary node in deoxygenated solution containing 0.1% (w/v) agar, 5.0 mM KCl and 0.50 mM CaSO442. The cylindrical platinum electrode was polarized relative to a silver/silver-chloride reference. Voltage and current were monitored and digitalized as previously described43; 12 − 15 cm adventitious roots, which emerged from 1 cm below the water surface, were used for the ROL measurement. The ROL measurements were taken along each root by positioning the center of the electrode at distances of 20 mm behind the root tip.
Root box -pin board root sampling method
To evaluate the root system development, the root box pinboard root sampling method, which minimized the loss and destruction of roots, was applied to the soybean root44,45. Seeds were sterilized using CruiserMaxx (Syngenta Japan) and were imbibed in vermiculite wetted with water for 3 d and 25 ℃ as described. Then, seedlings were transferred to the root box (40 cm × 24 cm × 2 cm) containing the granular soil (Nihon Hiryo Co., Ltd., Gunma, Japan; pH 5.8–6.5; 0.2 g N, 2.5 g P2O5, 0.2 g K2O, 0.2 g MgSO4 per 1 L soil; soil density 0.8 − 0.9 kg/L; granule diameter 0.5–3.0 mm). Plants were grown in a temperature-controlled, naturally lite phytotron (30°C during the day, 20°C at night) for 5 weeks in Nagoya, Japan between September and November 2021. The seedlings were grown under the drained soil condition for 2 weeks. After 2 weeks, seedlings in root boxes were moved to the large container for the waterlogging treatment and grown for 3 weeks. During waterlogging, the water level was maintained at 3 cm above the soil surface. After 3 weeks of treatment, the roots were collected by pinboard with 936 (39 × 24) nails. We separated the root system into three regions (Region Ⅰ: from water level to soil surface, Region Ⅱ: from the soil surface to 4 cm below the soil surface, and Region Ⅲ: more than 4 cm below the soil surface). The root length of each region was measured by a root scanner (Expression 12000XL 6.2, Epson, Nagoya, Japan) and WinRHIZO Pro LA2400 (Regent Instruments Inc, Quebec, Canada). After measurement, the roots were dried at 70°C for 3 days to determine the DW of the roots (Extended Data Fig. 10).