Plant materials
NaN3 mutant lines in the IR64 rice variety background were developed and self-pollinated for over 15 generations by the Taiwan Agricultural Research Institute (Wufeng, Taichung, Taiwan, ROC)40. Among the 1,888 mutant lines, the AZ1302 mutant was identified as exhibiting inhibited aerenchyma and iron plaque formation in adventitious roots. To map the gene responsible for these phenotypes, AZ1302 was crossed with Nipponbare, a polymorphic parent, to produce F1 progenies and an F2 mapping population. The early-flowering Kitaake cultivar41 was used for the generation of CRISPR–Cas9-induced mutants and promoter–GUS reporter lines. For the generation of overexpression lines, an AZ1302-like recombinant inbred line (Nipponbare × AZ1302 F6) predominantly exhibiting SNPs similar to Nipponbare was used.
Growth conditions
Seeds were disinfected with 50% sodium hypochlorite (J. T. Baker Inc.) for 15 min and subsequently cleaned three times with water. The disinfected seeds were kept on moistened filter paper and incubated in darkness at 30 °C for five days. Three days after germination, the seedlings were transferred to 1/2 Kimura B nutrient solution (pH 4.75)42. For the iron plaque induction assay, two-week-old seedlings were treated with 360 µM FeSO4·7H2O for one week, with the nutrient solution refreshed every alternate day. The formation of iron plaque was visualized as a reddish coloration after one week of excess Fe treatment.
To assess tillering ability, the plant materials were cultivated in a paddy field at Agricultural Experimental Station, National Chung Hsing University, Wufeng Township, Taichung, Taiwan. The fertilization rate was 125 kg ha−1 N, 75 kg ha−1 P2O5 and 50 kg ha−1 K2O. Seedlings were individually transplanted at a density of 30 plants per plot with a spacing of 30 cm × 15 cm.
Root tissue embedding, sectioning and imaging
The tissues were fixed in 3% (w/v) agarose, and 80-µm sections were prepared using a vibratome microtome (VT1200S, Leica Microsystem GmbH). To assess the aerenchyma of SL mutants, root segments were fixed in 4% (w/v) paraformaldehyde prepared in 1× PBS (pH 6.9) under vacuum at room temperature for 1 h, and the fixed roots were embedded in 5% (w/v) agarose. The cut sections were visualized under a light microscope, and the proportion of aerenchyma was quantified using ImageJ software (v.1.43u, US National Institutes of Health)43. For representative imaging of cell wall structures, sections were stained overnight with 0.1% (v/v) SR2200 (ref. 44). Confocal imaging was performed using a Zeiss LSM 710 inverted confocal microscope equipped with a 405-nm laser line for excitation; emission signals were collected between 430 and 500 nm to detect the blue fluorescence emitted by SR2200. To determine the length and density of root hairs, root segments from the apical, middle and basal regions were imaged at ×10 and ×20 magnifications, respectively. Measurements of root hair density and length were also performed using ImageJ.
Evaluation of ROL
Qualitative evaluation of ROL in the whole roots was performed with methylene blue, which becomes bluish in the presence of oxygen45. A 0.1% (w/v) agar was prepared, and 13 mg l−1 methylene blue was added to the cooled solution. The blue solution was decolourized by 130 mg l−1 sodium dithionite (Na2S2O4) (Merck KGaA) to a colourless solution. The solution was transferred into a clear plastic case with compartments measuring 4.5 cm long, 1 cm wide and 15 cm high. The plants were held in such a way that the root–shoot junction was approximately 2 cm below the surface of the solution. The roots were immersed in the prepared solution for 60 min at ambient temperature.
Gene mapping
DNA was isolated from both parental lines and the F2 population (n = 126) by employing the DNeasy Plant Mini Kit (Qiagen). The Affymetrix rice 44K SNP array, which includes 44,100 SNPs evenly distributed throughout the rice genome, with approximately one SNP every 10 kb, was employed for genotyping46. Genotypes were analysed using Axiom Analysis Suite through the Best Practice Workflow (Thermo Fisher)47.
QTL IciMapping v.4.2 with inclusive composite interval mapping was employed to identify the QTL associated with iron plaque formation48,49. Markers with over 10% missing data were omitted from further analysis. The remaining markers were categorized using a LOD score threshold of 3, ordered using the k-Optimality algorithm (LOD 2-OptMAP) and rippled with a window size of 5 Mb. Recombination frequencies were converted to centimorgans (cM) using the Kosambi function, and QTL were mapped with parameters set at a step size of 1.0 cM and a PIN value of 0.001. A 1,000-permutation test was performed to estimate the QTL LOD threshold at a 95% confidence level. The additive effects and phenotypic variance explained for each QTL were also calculated.
RT-PCR analysis
The extraction of total RNA from 21-day-old adventitious roots and third leaves was conducted using a Total RNA Mini Kit (Plant) (Geneaid Biotech Ltd). Complementary DNA was synthesized from the isolated RNA with iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.). RT-PCR was conducted to amplify full-length coding sequences, intron retention and exon skipping using specific primers. OsUBC2 served as the internal control for RT-PCR (Supplementary Data 1).
Exogenous carotenoid biosynthesis inhibitor treatment
Norflurazon and dichlormate were used to inhibit carotenoid biosynthesis through the inhibition of phytoene desaturase50 and ζ-carotene desaturase51, respectively. Stock solutions of 1 mM norflurazon (LGC Group) and 10 mM dichlormate (LGC Group) were prepared in DMSO. Two-week-old plants were treated with 0.1 µM norflurazon and 10 µM dichlormate, which were incorporated into 1/2 Kimura B solution. The visualization of aerenchyma and iron plaque was performed in three-week-old plants.
Knockout and overexpression assays
Four guide RNAs targeting the PSY2 gene were designed for generating CRISPR–Cas9 mutants. The oligonucleotide sequences for generating the single guide RNAs are provided in Supplementary Data 2. The CRISPR–Cas9 expression vector pYLCRISPR–Cas9Pubi-H was transformed into Agrobacterium tumefaciens EHA105 and subsequently transformed into calli derived from the japonica rice variety Kitaake (wild type). Three independent homozygous psy2 mutant lines free of the Cas9 transgene (T3 generation) were obtained, and the mutations were verified by sequencing using primer pairs flanking the target sites (Supplementary Data 1). Aerenchyma and iron plaque formation, ROL, root and shoot length, and root and shoot dry weight were assessed.
For the overexpression of PSY2, the entire coding DNA sequence of the PSY2 gene was amplified from Kitaake using RT-PCR. The pZmUBI::PSY2 expression vector was constructed in the backbone of vector pMHb7Fm21GW-UBIL and introduced into Agrobacterium. This construct was transformed into calli of Kitaake, CRISPR–Cas9-induced psy2 mutants and the Nipponbare × AZ1302 F6 recombinant line (a Nipponbare-like line carrying the PSY2 gene from AZ1302). Two independent transgenic lines exhibiting higher OsPSY2 expression were obtained, and aerenchyma and iron plaque formation was assessed.
Elemental analyses
The elemental concentration in iron plaque, roots, shoots and brown rice was assessed via ICP optical emission spectrometry (PerkinElmer Inc.). A DCB solution was applied to extract iron plaque surrounding the roots52. Roots after DCB extraction, shoots and grains were cleaned sequentially with ddH2O, 10 mM CaCl2 and ddH2O again, each for 20 min. After cleaning, the tissues were dried at 70 °C for three days. Approximately 1 ml of HNO3 was added to the weighed tissues in Teflon tubes and allowed to stand at room temperature overnight. The following day, 0.5 ml of H2O2 was added and held at room temperature for 30 min before microwave digestion using a MARS 6 PFAS Extraction system (CEM Corporation). After digestion, the solution was transferred into a 15-ml tube, and 8.5 ml of 2% HNO3 was added. The solution was filtered using 0.45-µm filters into new 15-ml tubes and stored until elemental analysis was conducted.
To visualize Fe in the roots, fresh roots from 28-day-old plants were carefully washed and dried using filter paper. Root segments taken at 3–4 cm from the tip were fixed in 2% agarose and cut into 200-μm transverse sections with a vibrating blade microtome (VT1200S, Leica Microsystem GmbH). These sections were mounted on double-sided tape affixed to microscope glass slides and set to dry at room temperature. The spatial scattering of 56Fe within the root sections was mapped using a quadrupole ICP mass spectrometer (Agilent 7800; Agilent Technologies), connected to a laser ablation system (NWR 266; ESI). Ablation was conducted with a Nd:YAG laser (266-nm wavelength), using a 5-µm spot size and a cycle frequency of 50 Hz. Argon carrier gas transported the ablated material to the ICP mass spectrometer. Data processing was performed using IOLITE v.3.65 software (Iolite Softwares Private Limited), and the 13C+ signal served as an internal normalization standard to mitigate signal variations caused by differences in tissue density.
Expression pattern analysis
The spatial distribution of PSY2 gene expression was assessed using a promoter–GUS reporter system. A 1,917-bp region upstream of the PSY2 start codon (ATG) was amplified via PCR using the Nipponbare cultivar’s genomic DNA (Supplementary Data 1). The amplified promoter region was cloned into the binary vector pGWB3 (ref. 53). A p35S::GUS construct served as a positive control. Both constructs were transformed into the Kitaake background.
Roots and shoots of transgenic plants aged three weeks were subjected to GUS staining using buffer (2 mM X-Gluc, 2 mM K3Fe(CN)6, 2 mM K4Fe(CN)6·3H2O, 50 mM NaHPO4 buffer (pH 7.2), 0.2% (v/v) Triton X-100) at 37 °C. The pPSY2::GUS transgenic plants were stained for up to two days, while the p35S::GUS plants were stained overnight. Chlorophyll was decolourized using 95% ethanol, and stained tissues were photographed. Cross-sections of the tissues were visualized and imaged under a light microscope.
Quantification of carotenoids in root tissues
For carotenoid extraction, 19-day-old plants were treated with 10 µM dichlormate for 48 h to partially inhibit β-carotene biosynthesis to obtain the quantification of phytoene as well as β-carotene51. Freeze-dried samples (100 mg) were homogenized using liquid nitrogen and transferred into 2-ml Eppendorf tubes. The powdered samples were mixed with 1 ml of ethanol containing 1 mg ml−1 butylated hydroxy anisole (Sigma Aldrich) to prevent the oxidation of carotenoids. After vortexing, the samples were sonicated on an ice bath for 15 min using Branson 3510 ultrasonic cleaner (Marshall Scientific) and centrifuged at 4 °C at 1,800 g for 5 min. After 200 µl of supernatant was collected, the extraction was repeated twice more, and 1 ml and 800 µl of supernatants were collected in the subsequent extraction. The collected supernatants were kept in darkness at −20 °C before further processing.
Carotenoids were identified and quantified using an Agilent 1290 Infinity II UPLC system (Agilent Technologies) coupled online to an atmospheric pressure chemical ionization source of an Agilent 6545 XT quadrupole time-of-flight mass spectrometer. The samples were separated using a YMC Carotenoid column (3 μm, 2.0 × 150 mm, YMC Co., Ltd). The column temperature was 30 °C. The mobile phases were methanol:water (9:1, v/v; eluent A) and isopropanol:methanol (7:3, v/v; eluent B). The flow rate was 0.3 ml min−1. The instrument was operated in positive full-scan mode (m/z of 100–1,500). Chromatogram acquisition, detection of mass spectral peaks and their waveform processing were performed using Agilent Qualitative Analysis v.10.0 and Agilent MassHunter Pro1finder v.B.10.00 software.
Quantification of ABA in root tissues
To determine ABA content, 100 mg of fresh root tissue was harvested in 2-ml Eppendorf tubes. Liquid nitrogen was employed to grind the samples into a fine powder, which was then mixed in 500 µl of extraction solution (2-propanol/H2O/HCl, 2:1:0.002) spiked with an internal standard (50 µl of methanol containing 2 ng of d6-ABA). The tubes were agitated for 30 min at 4 °C. Next, 1 ml of dichloromethane was added to each tube, followed by another round of agitation for 30 min at 4 °C. After mixing, the tubes were centrifuged at 13,000 g for 5 min at 4 °C, resulting in plant debris separating the two liquid phases. The lower phase (500 µl) was carefully collected and dried using Labconco CentriVap Benchtop Vacuum Concentrators (Fisher Scientific).
The dried extracts were dissolved in 100 µl of 80% methanol and subjected to vortexing for 5 min and then centrifugation at 13,200 g at 4 °C for 10 min. The resuspended samples were subjected to analysis using UPLC (Waters) coupled online to the Waters Xevo TQ-XS triple quadrupole mass spectrometer (Waters). The sample (in duplicate with 5 μl per injection) was separated with a Waters ACQUITY UPLC HSS T3 column (1.8 µm particle size, 2.1 × 100 mm). The column was operated at 30 °C with a flow rate of 0.3 ml min−1. The mobile phase comprised a gradient elution system of aqueous solution with 0.1% acetic acid and methanol with 0.1% acetic acid. Multiple reaction monitoring in negative mode was employed to monitor the characteristic MS transitions for ABA (m/z, 263 → 153) and d6-ABA (m/z, 269 → 159). Data were acquired and processed using MassLynx v.4.2 and TargetLynx software (Waters).
Quantification of SLs in root exudates and roots
SLs present in root exudates were collected and quantified using an established protocol24. Briefly, phosphorous deficiency treatment was conducted on two-week-old plants for seven days, and the media containing exudates were collected. Root exudates enriched with 2 ng of GR24 were loaded onto a preconditioned C18-Fast Reversed-SPE column 500 mg/3 ml (Grace) with 3 ml of methanol and subsequently 3 ml of water. The column was washed with 3 ml of water, followed by elution of SLs twice with 2 ml and 3 ml of acetone. The SL-containing fractions were concentrated to approximately 500 μl of an aqueous solution and then extracted with 1 ml of ethyl acetate. For LC-MS/MS analysis, the enriched SL organic fraction (750 μl) was vacuum-dried, resuspended in 100 μl of acetonitrile:water (25:75, v:v) and filtered through a 0.22-μm filter.
SLs from roots were extracted following the procedure described by Wang et al.14. Lyophilized and ground rice roots (25 mg) were spiked with 2 ng of GR24. Two separate extractions were performed with sonication using a Branson 3510 ultrasonic cleaner (Marshall Scientific), each using 2 ml of ethyl acetate for 15 min. Each extraction was followed by centrifugation at 1,800 g for 8 min at 4 °C. The obtained supernatants were pooled and vacuum-dried. The residual SLs were resuspended in 50 μl of ethyl acetate, mixed with 2 ml of hexane and loaded onto a 500 mg/3 ml silica SPE column (Grace). Then, 3 ml of hexane was passed through the column, and SLs were eluted with 3 ml of ethyl acetate. The eluates were vacuum-dried, resuspended in 150 μl of acetonitrile:water (25:75, v:v) and filtered through a 0.22-μm filter for LC-MS/MS analysis. Quantification of SLs was conducted on a UHPLC-Triple-Stage Quadrupole mass spectrometer (Thermo Scientific Altis) following established procedures54.
Bioassay for Striga hermonthica seed germination
Striga seed germination was assessed by following a published method55. Briefly, Striga seeds were preconditioned at 30 °C under moist conditions for ten days. 50 µl of root exudates collected from Kitaake and psy2 mutants were treated with Striga seeds. Subsequently, the seeds were incubated at 30 °C in the dark for another two days. The germinated and non-germinated seeds were counted with a binocular microscope, and the germination rate was determined.
Exogenous ABA and GR24 treatment
Stock solutions of 25 mM (rac)-GR24 (PhytoTech Labs, Inc.), 10 mM (+)-GR24 (US Biological) and 250 µM ABA (Merck KGaA) were prepared in DMSO. For treatment, 11-day-old hydroponically grown plants were transferred to 1/2 Kimura B solution containing 0.01 µM ABA or 5 µM GR24, with DMSO serving as the control. The treatments were continued for ten days. For iron plaque induction, excess iron treatment was done on 14-day-old plants as mentioned previously. Aerenchyma and iron plaque formation was observed in two-week-old and three-week-old plants. The DCB solution was applied to extract iron plaque surrounding the newly emerged adventitious roots. Root dry weight, root length, shoot length, root hair density and root hair length were also measured.
Genetic and chemical disruption of the SL and ABA pathways
The SL biosynthesis mutants d17 and d10, with disruption in the conversion from 9-cis-β-carotene to carlactone56, and the SL signalling mutant d14 (ref. 57) were used to assess the role of SLs in aerenchyma and iron plaque formation. The wild-type Shiokari and the mutants were grown for three weeks, and aerenchyma in the adventitious roots was visualized at 2–3 cm and 4–5 cm from the root tips. To determine the function of the ABA pathway in iron plaque formation, the specific ABA signalling inhibitor AA1 was applied15. Wild-type (Kitaake) plants were treated with 1 µM AAl at the ten-day-old stage, and excess Fe treatment was applied to two-week-old plants. The iron plaque phenotype was examined at the three-week-old stage.
Data analysis and visualization
If not otherwise stated, the data were analysed and visualized using R v.4.1.3 (ref. 58). The normality of the data and the homogeneity of their variance were determined via the Shapiro–Wilk test and Levene’s test, respectively. P values were calculated for two-tailed t-tests. For multiple comparisons between genotypes and treatments, two-way analysis of variance followed by Tukey’s post hoc test was conducted. For data not meeting the assumptions of normality and/or homogeneity of variance, Welch’s t-test and the Kruskal–Wallis test followed by the Dunn test were conducted.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
