X gluc

The lamina joints of OsMYB7-OE1 carrying a β-glucuronidase (GUS) reporter gene driven by the OsMYB7 promoter (Supplementary Figure S3A) were subjected to histochemical GUS staining according to the previously described method (Jefferson et al., 1987 (link)) with some modifications. Lamina joints at developmental stages S3, S4, and S5 were sampled and vacuum-infiltrated with a X-Gluc reaction buffer containing 100 mM sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 1 mM X-Gluc (Sigma-Aldrich, Saint Louis, MO, USA) for 30 min in darkness, followed by overnight incubation at 37°C under dark conditions. The GUS histochemical staining buffer was then replaced with 70% (v/v) aqueous ethanol to remove chlorophylls. After complete decolorization, images of lamina joints were capture with a digital camera, and transverse hand-cut sections of lamina joints were subsequently photographed using a stereo microscope (SteREO Discovery.V12, Carl Zeiss Microscopy GmbH, Jena, Germany) with dark filter.

The β-glucuronidase (GUS) activity was measured as described previously [27 (link)] and used to analyze the expression of the M2H gene promoter. Seeds were placed in GUS staining solution containing 50 mM sodium phosphate (pH 7.0), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.1% Triton X-100, and 1 mM X-Gluc (Sigma-Aldrich), and then incubated at 37 °C for 24 h. The stained embryos were immersed in 70% ethanol for several hours, and then microscopic analyses were conducted using an SZ51 stereomicroscope (Olympus).