| Growth Protocol: |
For plate experiments, seeds were dehulled and surface sterilized in 50% (v/v) bleach solution for 30 min, rinsed ten times with sterile distilled water and grown on plates (100 cm2) containing 0.5x Murashige and Skoog medium (MS), 1% (w/v) agar 1% (w/v) sucrose for 7 days (16h day / 8h night; at 28°C/25°C day/night; 110 μEm-2s-1). For greenhouse experiments, seeds were imbibed for 2 d in 5% (v/v) Liquid SmokeTM (Colgin) at 28°C in darkness to promote consistent germination. The seeds were then rinsed with distilled water and returned to 28° C in darkness for 24 h. Germinated seeds (21) were transferred to pots (15 cm diameter and 18 cm height), filled with Profile® Greens GradeTM and placed in a greenhouse at the University of California, Riverside California during June and July 2016. Temperature was controlled at 28° C for 16 h per day and at 25° C for 8 h per night. Pots were placed in trays and watered everyday with fresh water (depth of 1 cm water tray). After 10 d of growth, irrigation was with fertilizer water (Peters® Excel: 21-5-20 at 100 ppm N). Each pot contained 21 plants (combined as a single replicate). The location of each pot was randomized in multiple trays/tanks used. After 21 d of development (from imbibition), when plants were at the V4 to V5 growth stage, four water regimes were applied. For water-sufficiency (control), pots were maintained by daily irrigation with fertilizer water. For water deficit (drought), pots were removed from the tray and watering was stopped. For waterlogging, pots were placed in tanks (56-qt clear storage box; Sterilite® 1659) and flooded with water to 2-3 cm above the root/shoot junction. For submergence, at solar midday, pots were transferred to tanks (grey plastic Rubbermaid® FG265500 containers) and fully submerged with water, with the flood line 70 cm above the root/shoot junction. The containers were covered with two layers of shade cloth (SHANS 90% UV Block). For the field experiments, seedlings were germinated in Magenta boxes on selective medium after sterilization, transferred to Profile®, and grown for 14 d in the greenhouse before before transplantation into a pre-fertilized paddy at the Agricultural Operations field at the University of California Riverside Agricultural Experiment Station. The paddy was maintained at 8-10 cm depth for 35 d. Planting of genotypes was based on a randomized design with ten plants per biological replicate. |
| Treatment Protocol: |
For plate experiments, Oryza sativa cv. Nipponbare seeds from transgenic lines were dehulled and surface sterilized in 50% (v/v) bleach solution for 30 min, rinsed ten times with sterile distilled water and grown on plates (100 cm2) containing 0.5x Murashige and Skoog medium (MS), 1% (w/v) agar 1% (w/v) sucrose for 7 days (16h day / 8h night; at 28°C/25°C day/night; 110 μEm-2s-1). For greenhouse experiments, seeds were imbibed for 2 d in 5% (v/v) Liquid SmokeTM (Colgin) at 28°C in darkness to promote consistent germination. The seeds were then rinsed with distilled water and returned to 28° C in darkness for 24 h. Germinated seeds (21) were transferred to pots (15 cm diameter and 18 cm height), filled with Profile® Greens GradeTM and placed in a greenhouse at the University of California, Riverside California. Temperature was controlled at 28° C for 16 h per day and at 25° C for 8 h per night. Pots were placed in trays and watered everyday with fresh water (depth of 1 cm water tray). After 10 d of growth, irrigation was with fertilizer water (Peters® Excel: 21-5-20 at 100 ppm N). Each pot contained 21 plants (combined as a single replicate). The location of each pot was randomized in multiple trays/tanks used. After 21 d of development (from imbibition), when plants were at the V4 to V5 growth stage, four water regimes were applied. For water-sufficiency (control), pots were maintained by daily irrigation with fertilizer water. For water deficit (drought), pots were removed from the tray and watering was stopped. For waterlogging, pots were placed in tanks (56-qt clear storage box; Sterilite® 1659) and flooded with water to 2-3 cm above the root/shoot junction. For submergence, at midday, pots were transferred to tanks (grey plastic Rubbermaid® FG265500 containers) and fully submerged with water, with the flood line 70 cm above the root/shoot junction. The containers were covered with two layers of shade cloth (SHANS 90% UV Block). For the field experiments, seedlings were germinated in Magenta boxes on selective medium after sterilization, transferred to Profile®, and grown for 14 d in the greenhouse before before transplantation into a pre-fertilized paddy at the Agricultural Operations field at the University of California Riverside Agricultural Experiment Station. The paddy was maintained at 8-10 cm depth for 35 d. Planting of genotypes was based on a randomized design with ten plants per biological replicate. Five independent biological replicates were grown for each treatment. Pots were placed in trays and watered everyday with fresh water (depth of 1 cm water tray). After 10 d of growth, irrigation was with fertilizer water (Peters® Excel: 21-5-20 at 100 ppm N). Each pot contained 21 plants (combined as a single replicate). The location of each pot was randomized in multiple trays/tanks used. After 21 d of development (from imbibition), when plants were at the V4 to V5 growth stage, four water regimes were applied. For water-sufficiency (control), pots were maintained by daily irrigation with fertilizer water. For water deficit (drought), pots were removed from the tray and watering was stopped. For waterlogging, pots were placed in tanks (56-qt clear storage box; Sterilite® 1659) and flooded with water to 2-3 cm above the root/shoot junction. For submergence, at solar midday, pots were transferred to tanks (grey plastic Rubbermaid® FG265500 containers) and fully submerged with water, with the flood line 70 cm above the root/shoot junction. The containers were covered with two layers of shade cloth (SHANS 90% UV Block). A fiber optic oxygen meter (Neofox Sport, Ocean Optics, Dunedin, USA), in five replicates was used to determine the percentage of dissolved oxygen in the submergence tanks was measured at the start and end of the experiment. Oxygen content was stable a 18.90 ± 0.03% at the start (0 d) and end of the experiment. |
| Extract Protocol: |
TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: tissue samples were increased to isolate polysomes from low abundant cell populations; ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (Townsley et al 2014). Random primer primed RNA-seq libraries were prepared from poly(A)+ selected mRNA as described. (Townsley et al 2014, Reynoso et al. 2019). Nuclei were purified from frozen and pulverized tissue as previously described for O. sativa (Reynoso et al., 2018) with minor modifications including the use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract and extend centrifugation times. Tissue was resuspended in an ice-cold mortar containing 10 mL of freshly prepared nuclei purification buffer (NPB: 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, pH, 7.0) containing 200 uL Protease Inhibitor Cocktail (0.4X, Sigma, P9599) per 50 mL of buffer. The homogenized extracts were filtered through a 30 µM nylon mesh to remove cell debris and centrifuged at 1000 x g for 15 min at 4° C to pellet nuclei. Nuclei were resuspended in 1 mL of NPB and 25 µL of M-280 streptavidin-coated Dynabeads (Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated in a cold room at 4° C for 30 min. The nuclei/beads suspension was diluted to 14 mL with NPB supplemented with 0.1% (v/v) Triton X-100 (NPB-T), in a 15 mL Falcon tube, mixed thoroughly and placed in a 15 ml magnet (adapted NEB 50 mL tube magnet) to capture bead-bound nuclei for 1 min at 4° C. The supernatant was carefully removed using a plastic Pasteur pipette, taking care to remove bubbles to avoid disturbing the beads. Beads were resuspended in 14 ml of cold NPB-T, placed on a rotating mixer for 30 sec, and then placed back in the 15 ml magnet to capture the beads-nuclei at 4° C for 1 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube |
| Library Construction Protocol: |
Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using 20,000-50,000 nuclei as previously described (Maher et al 2017, Reynoso et al., 2019), with slight modifications. Minor modifications in nuclei purification include: 1) the use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract, 2) extended centrifugation times (Reynoso et al 2018b, Reynoso et al 2018), and 3) using AMPureXP beads instead of columns to purify amplified libraries. TRAP was performed as previously described (Mustroph et al., 2009, Reynoso et al., 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (5). Total RNA was extracted from frozen tissue using polysome extraction buffer (Mustroph et al 2009) followed by LBB polyA mRNA isolation using biotinylated oligodT and streptavidin magnetic beads (Townsley et al 2014). Random primer-primed RNA-seq library construction for polyadenylated total RNA and polyadenylated TRAP RNA was performed according the BrAD-seq method (Townsley et al 2014) in at least three biological replicates for each condition. |
