Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings

Abstract

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.

Figure 1

Accumulation of phyA in Arabidopsis seedlings in the dark. A, From 24 to 144 h after the start of induction of germination by 24 h of white light, Ptot was determined by in vivo spectroscopy in phyB-5 (●) and phyA-201 phyB-5 (○). B, Samples of etiolated seedlings of phyB-5 and phyA-201 phyB-5 were analyzed by immunoblotting of 25 μg of protein and probing with an antiserum against phyA. All subsequent experiments were performed with phyB-5 only.

Figure 2

Destruction of phyA in continuous light. Three-day-old etiolated seedlings were exposed to continuous red light (A and C) or far-red light (B and D). At regular time intervals Ptot was determined by in vivo spectroscopy (A and B) or samples were analyzed by immunoblotting of 25 μg of protein and probing with an antiserum against phyA (C and D).

Figure 3

Kinetic analysis of the destruction of phyA in continuous light. Data of Figure 2, A and C, and of similar destruction kinetics at 718, 692, and 436 nm were fitted to a first-order time law. k_D,obs was plotted against ϕ.

Figure 4

Destruction and dark reversion of phyA after a red-light pulse. Following a red-light pulse of 5 min and retransfer into darkness (23°C), Ptot (●) and Pfr (□) were determined in 3-d-old etiolated seedlings by in vivo spectroscopy. Pr (▪) was calculated as difference between Ptot and Pfr. Data were fitted to a first-order time law (lines). Destruction (decrease of Ptot) proceeded with a half-life of 20 min. The decrease in Pfr (destruction plus dark reversion) paralleled the decrease of Ptot exactly. There was no dark reversion (increase in Pr) detectable. Data points represent the means of six parallels for Pfr-Pr pairs and means of eight parallels for Ptot values. Error bars indicate ses.

Figure 5

Levels of phyA mRNA after irradiation and subsequent reaccumulation of phyA. After 2 h of red light (lane 1), 2 h of darkness (lane 2), 6 h of far-red-light (lane 3), 6 h of darkness (lane 4), or 6 h of white light (lane 5), 3-d-old etiolated seedlings were harvested and total RNA was extracted. Blots were hybridized with a probe against Arabidopsis phyA (A), and reprobed with an Arabidopsis 18 S rRNA as a loading control (B). Following 2 h of red light (●) or 6 h of far-red light (○) and retransfer into darkness, Ptot was determined by in vivo spectroscopy in 3-d-old etiolated seedlings (C).

Figure 6

Pfr-induced Pr destruction. Following a red-light pulse of 5 min and retransfer into darkness, Ptot was determined in 3-d-old etiolated seedlings by in vivo spectroscopy (○, replot of data from Fig. 3). Alternatively, 15 min after the red-light pulse, a far-red-light pulse of 5 min was applied. The time course of Ptot in subsequent darkness was measured (▪). Similarly, the time course of Ptot after the far-red-light pulse alone was measured (●). The ordinate was shifted by 20 min to have the end of the initial red light pulse at −20 min for both curves and the end of the reverting far-red-light pulse at 0 min. Data points after 0 min were fitted to a first-order time law (lines). Destruction of Pfr (○) and Pr (▪) proceeded with half-lives of 20 and 10 min, respectively. The amplitude of Pr destruction (17%) was one-half of the amplitude of Pfr destruction (36%) after 20 min.

Figure 7

Model of phyA destruction in continuous light. A, Schematic representation of the employed two-step model of destruction (for details, see text). B, Data of destruction kinetics (Fig. 3) were fitted according to the model in Figure 7A. The apparent rate constant k_3,app was plotted against ϕ; k_D was 2.53 × 10⁻² min⁻¹.