Plant material construct development and growth conditions
The background/wild type of osarf1, OE-ARF1, cesa6, arf1cesa6 and NG-CESA6 in this study was rice (Oryza sativa ssp. japonica) variety 9522. Nipponbare (Nip), ein2 and eil1eil2 were sourced from the group of R. Huang. The osarf1, cesa6 and arf1cesa6 knock out alleles were generated using CRISPR–Cas9 technology, with mutant genotyping conducted through PCR amplification of the target regions followed by sequencing of the products. We amplified OsARF1 coding (2,100 bp) and promoter (2,915 bp in front of ATG) sequences and then cloned them into pTCK303 and pCAMBIA1301 to produce OE-ARF1 and ProOsARF1:GUS, respectively. We also amplified OsCESA6 promoter (2,877 bp upstream of ATG) sequence and then cloned the segment into pCAMBIA1301 to produce ProOsCESA6:GUS. mNeonGreen was fused to the OsCESA6 genomic sequence under the control of the OsCESA6 promoter and was cloned into pCAMBIA1301 to create NG-CESA6, as described in reference15. All plasmids were constructed using the In-Fusion HD Cloning Kit (Takara) and were verified by sequencing at BGI, Beijing, China. These plasmids were introduced into the calli of 9522 or cesa6 via Agrobacterium (EHA105)-mediated transformation, with hygromycin B used for selection as outlined in reference32. All primers used are listed in Supplementary Table 1.
All rice samples were grown and collected at the paddy field of Shanghai Jiao Tong University, under natural conditions from June to October. Seedlings were cultivated in a plant incubator at 28 °C with a 16 h:8 h light:dark cycle and 50%–70% humidity.
Soil compaction details
Loamy sand from the Newport series (comprising 83.2% sand, 12.1% clay and 4.7% silt; with 2.93% organic matter and a pH of 6.35; classified as FAO Brown Soil) and clay soil were collected from the University of Nottingham farm at Bunny, Nottinghamshire, UK (52.52° N, 1.07° W). Both soils were passed through a 2-mm sieve. The moisture content was determined by drying the soil at 45 °C until a constant weight was achieved.
Mesocosms were prepared by packing them with the sieved soil to achieve a bulk density of 1.2 g cm−3, to simulate a non-compacted condition, and 1.6 g cm−3 for a compacted condition. Rice seeds were sterilized with a 25% bleach solution for 10 min and subsequently rinsed six times with sterilized water. The seeds were then placed on filter paper for 48 h in a dark incubator chamber to allow for germination. One germinated seedling per mesocosm was positioned on the soil surface and covered with a 1 cm top layer of soil at 1.2 g cm−3, in both non-compacted and compacted soil conditions. The seedlings were then placed in a rice growth chamber, which was maintained at 28 °C, with a 12 h photoperiod and 70% relative humidity.
X-ray CT imaging
Five-day-old seedlings of wild type, osarf1 and cesa6 were grown in 3D-printed columns (33 mm in diameter and 100 nm in height) filled with sandy loam soil under non-compacted (1.2 g cm−3) and compacted (1.6 g cm−3) conditions, respectively. They were then imaged using a GE Phoenix v|tome|x M 240 kV X-ray tomography system at The Hounsfield Facility, University of Nottingham. Three-dimensional image reconstruction was carried out using Datos|REC software (GE Inspection Technologies). The roots were segmented from the soil using a polyline tool in VGStudioMAX V2.2 (Volume Graphics) to demonstrate the root length phenotype. The scanning protocol involved collecting 3,240 projection images in FAST mode (continuous rotation), with the X-ray tube set to an energy of 140 kV and a current of 200 μA. The detector’s exposure time was 131 ms, and the voxel resolution was 57 μm. Each scan had a duration of 7 min.
Agar experimental details
Agar at concentrations of normal (0.3%) and dense (0.6%) (Sigma Aldrich 7002) was boiled and poured into tanks measuring 7.5 cm in diameter and 10 cm in length to simulate non-compacted and compacted soil conditions, respectively. After the agar solidified, germinated seeds were placed on the media surface, followed by watering (1 cm height). The seeds were then grown in a plant incubator (28 °C, 16 h light:8 h dark, 70% relative humidity) for 5 days. Finally, root phenotypes were imaged using a Nikon camera, and the images were analysed with ImageJ software.
... continue reading