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