Figure 5.

C. hirsuta Seedlings Have Higher phyA Levels Than Those of Arabidopsis.

(A) Cartoon showing the design of the experiment. At^WT and Ch^WT, grown as in Figure 1A, were harvested at the indicated times of W or W+FR treatments (asterisks) for RNA extraction.

(B) Evolution of PHYA transcript levels in At^WT and Ch^WT seedlings grown as detailed in (A). Primers used (Supplemental Figure 8A) allow quantifying and comparing expression levels by RT-qPCR between both species. PHYA transcript abundance was normalized to three reference genes (EF1α, SPC25, and YLS8). Values are means and se of three independent RT-qPCR biological replicates relative to PHYA transcript levels of day-3 Arabidopsis seedlings. Two-way ANOVA showed that PHYA levels are significantly different (**, P < 0.01) between species under either W or W+FR.

(C) Immunoblot detection of phyA and tubulin with the antibodies indicated in Figure 3C in extracts of At^WT and Ch^WT seedlings grown as detailed at the top of the panel: 5-d-old etiolated seedlings were exposed to W light, and material was harvested before and after 6 h of W exposure (arrows).

(D) Evolution of relative phyA protein levels (PHYA:TUB) in At^WT and Ch^WT seedlings exposed to simulated shade, as detailed at the top of the panel: 5-d-old etiolated seedlings were exposed to W+FR light, and material was harvested before and after 6, 8, and 10 h of simulated shade exposure (arrows). Values are means and se of four independent biological replicates relative to PHYA:TUB levels of etiolated At^WT seedlings. Two-way ANOVA showed that relative PHYA levels under W+FR are significantly increased (**, P < 0.01) in C. hirsuta over Arabidopsis.