Histochemical GUS staining, GFP, and fluorescence resonance energy transfer (FRET) experiments
Histochemical GUS staining, GFP microscopy, and FRET were performed as previously described with some modifications (Datta et al., 2006; Hou, Wu, & Gan, 2013; Zhang, Zhang, et al., 2014). For the GUS-staining assay, the young seedlings (4 d after germination) were fixed and incubated in GUS-staining solution for 24 h at 37 °C. The stained samples were then cleaned with 75% ethanol and observed under a dissecting microscope.
For the GFP assay, the fusion COL13 -GFP constructs were transformed into protoplasts for transient expression as previously described (Wu et al., 2009). Ten stable transgenic plants with COL13 -GFP were obtained using the floral-dip method. Photographs of GFP were taken using a confocal microscope Olympus.
For the FRET assay, images were acquired using an Olympus confocal microscope, and protoplasts were visualized 16 h after transformation. The CFP was excited by a laser diode 405 laser and YFP, by an argon-ion laser. The target regions were bleached with 100 iterations using an argon-ion laser at 100% power.