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; Lee et al., 2007; Peter H Quail, 2002; von Arnim & Deng, 1994), we investigated the expression of COL13 in the absence ofPHYB , 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 of the WT, whereas the expression of COL13 in the cop1 mutants was increased (Fig. 3a, b). As the expression of COL13 decreased the most in the col3 mutant, we generated transgenic lines expressing GUS under the control of the COL13 promoter with the col3mutant background. Interestingly, although the COL13 promoter was active in the hypocotyls and cotyledons in the 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 was in Col-0 and col3 was in the WS background, crossing lines from different backgrounds would likely affect hypocotyl length. To reduce the effect of the background, we used the F1 hybrid of Col-0× WS as the WT. We found that, although hypocotyl length in the double-mutant col13 col3 was longer than in the WT seedlings, it was not significantly different from hypocotyl length in the single mutants, col13 or col3. (Fig. 3d). To confirm this result, we created the RNAi lines of COL13 in the col3 mutant background (Fig. 3e), and we obtained the same result as in Fig. 3d. Additionally, we also generated a COL13 -OX line in thecol3 mutant background and showed that the hypocotyl length in this strain was similar to that of the WS and significantly shorter than that of the col3 mutant (Fig. 3e). In other words,COL13 overexpression rescued the phenotype exhibited by thecol3 mutant. Taken together, our results suggest thatCOL13 might be downstream of COL3 in the red-light-mediated signaling pathway.