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
RT-PCR and qRT-PCR analysis revealed that the expression of COL13in 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.