Genetic interaction and physiological characterization of hypocotyl elongation
Given that the phyB , hy5, col3 and cop1 mutations can affect hypocotyl elongation under red light conditions(Datta et al., 2006; J. Lee et al., 2007; Peter H Quail, 2002; von Arnim & Deng, 1994), we investigated the expression of COL13 in the absence of PHYB ,COL3 , HY5 and COP1 . Semi-quantitative PCR and quantitative PCR (qPCR) analysis revealed that the expression ofCOL13 in phyB , col3 or hy5 knockout plants was significantly reduced compared with that in the WT, while the expression of COL13 in the cop1 mutants was increased (Fig. 3a,b). As the expression of COL13 decreased the most in thecol3 mutant, we generated transgenic lines expressing GUS under control of the COL13 promoter in the col3 mutant background. Interestingly, while the COL13 promoter was active in the hypocotyl as well as cotyledon in WT seedlings, GUS expression was not detected in the hypocotyl in the col3 mutant background (Fig. 3c).
To understand the functional relationship and genetic interaction between COL13 and COL3 and their role in the regulation of hypocotyl growth, we generated a col13 ×col3 double mutant, and examined hypocotyl length under red light conditions. Given thatcol13 is in Col-0 and col3 is in WS background, crossing lines from different backgrounds very likely will have an effect on the hypocotyl length. To reduce the effect from background, we use the F1 hybrid of Col-0× WS as WT. We found that, while hypocotyl length in the double mutant col13×col3 was longer than in WT seedlings, it was not significantly different to hypocotyl length in the single mutants, col13 or col3. (Fig. 3d). To confirm this result, we made the RNAi lines of COL13 in the col3 mutant background (Fig. 3e), and we obtained the same result as Fig. 3d. Additionally, we also generated a COL13 -OX line in thecol3 mutant background, and showed that hypocotyl length in this strain was similar to that of WS, and significantly shorter than that of the col3 mutant (Fig. 3e). In other words, overexpression of COL13 rescued the phenotype exhibited by the col3mutant. Taken together, our results suggest that COL13 might be the downstream of COL3 in the red-light mediated signaling pathway.