X gluc

GUS assay was performed as described previously^{70 (link)} with minor modifications. Briefly, seedlings were grown in 24-well plates containing liquid LS medium supplemented with 0.5% sucrose under 16 h/8 h day/night cycle in a Percival plant growth chamber at 22 °C under a light intensity of 50 μmol m⁻² s⁻¹. Plants were inoculated at day 12 with bacterial strains. Bacterial strains were grown on R2A plates at 22 °C for 3 d, resuspended in 10 mM MgCl₂ and added to seedlings in LS medium without sucrose at OD₆₀₀ of 0.002. After treatment with SynCom strains for 5 h, seedlings were rinsed with 0.5 ml 50 mM sodium phosphate buffer (pH 7) and submerged in 0.5 ml GUS staining solution (50 mM sodium phosphate (pH 7), 0.5 mM K₄[Fe(CN)₆], 0.5 mM K₃[Fe(CN)₆], 1 mM X-Gluc (GoldBio, G1281C) and 0.01% Silwet-L77 (Bioworld, 30630216)). After vacuum infiltration for 10 min, plates were incubated at 37 °C overnight. Plants were fixed with a 3:1 ethanol:acetic acid solution at 4 °C for 1 d followed by transfer to 95% ethanol.

Gynoecia were dissected and pre-fixed with cold acetone for 20 min, rinsed, and transferred into GUS substrate solution: 50 mM sodium phosphate pH 7, 5 mM K3/K4 FeCN, 0.1% (w/v) Triton X-100, and 2 mM X-Gluc (Gold BioTechnology, Inc). After application of vacuum for 5 min, SPT::GUS and ARR16::GUS samples were incubated at 37°C for 12 hrs.

GUS staining of plant tissues was performed at 37 ^oC in a solution containing 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM K₄Fe[CN]₆, 0.5 mM K₃Fe[CN]₆, 0.1% triton X-100, and 1 mM X-Gluc (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium salt) (Gold Biotechnology). GUS-stained tissues were incubated in 70% ethanol to remove chlorophyll and cleared in the Hoyer's solution. GUS expression patterns were imaged with a Leica MZ FLIII fluorescent dissecting microscope using the imaging software Leica Application Suite V4.0.

Seedlings, leaves, flowers, siliques, and seeds without the seed coat were incubated overnight in a GUS staining solution [100 mM phosphate buffer (pH 7.0), 1 mM X-gluc (Gold biotechnology, St. Louis, MO), 10 mM EDTA, 0.1 % (v/v) Triton X-100] at 37 °C. Tissue samples were then treated with 95% (v/v) ethanol to remove chlorophyll and photographed under a microscope (Nikon Stereo Zoom).

For GUS staining, cotyledons or 4-week-old leaves were collected and infiltrated in 0.5 mg/ml X-gluc (Gold Bio COM), 50 mM sodium phosphate (pH 7.0), 10 mM EDTA buffer, followed by incubation at 37 °C overnight. Leaves were cleared in ethanol.

Gynoecia of different developmental stages were dissected and pre-fixed with cold acetone for 20 min, then rinsed and transferred into GUS substrate solution: 50 mM sodium phosphate pH 7, 5 mM K3/K4 FeCN, 0.1% (w/v) Triton X-100, and 2 mM X-Gluc (Gold BioTechnology Inc.). After application of vacuum for 20 min, all samples were incubated at 37°C for 96 h.

For detection of β-glucuronidase (GUS) activity, transverse leaf and root sections from plants in tissue culture or pollen grains from soil-grown plants were incubated in X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid; Gold Biotechnology, Inc., St. Louis, MO, USA) solution
[41 (link)] at 37°C for 24 or 48 hours.