Arabidopsis phytochrome a is modularly structured to integrate the multiple features that are required for a highly sensitized phytochrome

Abstract

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.

Figure 1.

Preparation of Transgenic Arabidopsis Lines Expressing phyA/phyB Chimeric Proteins. (A) Diagram of phyA and phyB. White and gray boxes indicate the phyA and phyB sequences, respectively. N, N-terminal extension (1 to 78 in phyA; 1 to 102 in phyB); PAS, PAS domain (79 to 185 in phyA; 103 to 219 in phyB); GAF, GAF domain (218 to 402 in phyA; 252 to 433 in phyB); PHY, PHY domain (413 to 593 in phyA; 444 to 624 in phyB). The four small rectangles indicate the chromophores. (B) Diagram of 16 phyA/phyB chimeric proteins. The phyA and phyB molecules were divided into four parts, and their respective sequences were shuffled between phyA and phyB. Numbers shown on the AAAA and BBBB sequence denote the amino acid positions of the borders. The four rectangles indicate the chromophores. (C) Immunoblot detection of the phyA/phyB chimeric phytochromes with a mouse monoclonal anti-GFP antibody in etiolated seedlings of representative transgenic lines. Five micrograms of crude protein extract was loaded in each lane.

Figure 2.

Hypocotyl Lengths of Arabidopsis Seedlings Expressing phyA/phyB Chimeric Proteins in the Dark. (A) Hypocotyl lengths of seedlings grown in the dark for 5 d. Data are the means ± se (n = 25). Ler, Landsberg erecta. (B) The effects of an FR pulse on the dark phenotype. Schematic representations of the growth conditions (top) and the hypocotyl lengths of seedlings (bottom) are shown. Seeds were irradiated with white light for 1 h to synchronize germination and then kept in the dark for 11 h. To eliminate residual Pfr, seeds were treated with a 5-min FR pulse (18 μmol/m²/s) and returned to the dark. Hypocotyl lengths were determined after 4.5 d (n = 25; mean ± se). WL, white light. (C) The relationship between hypocotyl length in the dark (ordinate) and protein expression levels (abscissa) in independent transgenic lines (see Supplemental Figure 2 online for enlarged views). Expression levels were estimated by densitometric analysis of the immunoblots (see Supplemental Figure 1 online) and are expressed in units relative to endogenous phyA. Data are the means ± se (n = 25). Asterisks indicate the lines chosen as representative, as shown in Figure 1. WT, the wild type.

Figure 3.

Subcellular Localization of phyA/phyB Chimeric Phytochromes in the Dark under Continuous FR (18 μmol/m²/s) or under Continuous R (5.5 μmol/m²/s). Three-day-old, dark-grown seedlings were treated with FR or R for 24 h before observation. The epidermis in the hook regions of seedlings was observed using a confocal laser scanning microscope. Arrowheads indicate the nuclei. D, dark. Bars = 10 μm.

Figure 4.

Hypocotyl Lengths of Arabidopsis Seedlings Expressing phyA/phyB Chimeric Proteins under Continuous FR. (A) Hypocotyl lengths of seedlings grown under continuous FR (18 μmol/m²/s) for 5 d. The hypocotyl lengths are presented relative to the dark (D). Data are the means ± se (n = 25). Ler, Landsberg erecta. (B) The relationship between hypocotyl length under continuous FR (ordinate) and protein expression levels (abscissa) in independent transgenic lines (see Supplemental Figure 5 online for enlarged views). Expression levels were estimated by densitometric analysis as for Figure 2C and are expressed in units relative to endogenous phyA. The hypocotyl lengths are presented relative to dark. Data are the means ± se (n = 25). Asterisks indicate the lines chosen as representative, as shown in Figure 1. WT, the wild type.

Figure 5.

Degradation of phyA/phyB Chimeric Proteins under Continuous R. (A) Immunoblot detection of the phyA/phyB chimeric proteins in seedlings treated with continuous R. Three-day-old, dark-grown seedlings were kept in the dark (D) or exposed to R (8.5 μmol/m²/s) for 24 h. The blots were probed with anti-GFP monoclonal antibodies (top). Five micrograms of total protein was loaded in each lane. To confirm equal protein loading, the same blots were subjected to Coomassie blue (CBB) staining (bottom). (B) Expression levels were estimated by densitometric analysis of the immunoblots using a dilution series. The data are presented relative to the dark levels. Data are the means ± se (n = 3).

Figure 6.

Hypocotyl Lengths of Arabidopsis Seedlings Expressing Chimeric Phytochromes under R. (A) Hypocotyl lengths of seedlings grown under R (3.3 μmol/m²/s) for 5 d. The hypocotyl lengths are presented relative to the dark (D). Data are the means ± se (n = 25). Ler, Landsberg erecta. (B) The relationship between hypocotyl length under R (ordinate) and protein expression levels (abscissa) in independent transgenic lines (see Supplemental Figure 8 online for enlarged views). Expression levels were estimated by densitometric analysis as for Figure 2C and are expressed in units relative to endogenous phyB. The hypocotyl lengths are presented relative to D. Data are the means ± se (n = 25). WT, the wild type.

Figure 7.

Hypocotyl Lengths of Arabidopsis Seedlings Expressing Chimeric Phytochromes under Weak R. (A) Hypocotyl lengths of seedlings grown under weak R (0.005 μmol/m²/s) for 5 d. The hypocotyl lengths are presented relative to the dark (D). Data are the means ± se (n = 25). Ler, Landsberg erecta. (B) The relationship between hypocotyl length under weak R (ordinate) and protein expression levels (abscissa) in independent transgenic lines (see Supplemental Figure 9 online for enlarged views). Expression levels were estimated as for Figure 2C and are expressed in units relative to endogenous phyB. The hypocotyl lengths are presented relative to the dark. Data are the means ± se (n = 25). WT, the wild type. (C) Fluence rate response curves for the inhibition of hypocotyl elongation under continuous R (cR). Seedlings were grown for 5 d under various fluence rates of R. The hypocotyl lengths are presented relative to D. Data are the means ± se (n = 25).

Figure 8.

The Structural Basis for Each phyA-Specific Function. N, N-terminal extension (1 to 78); PAS, PAS domain (79 to 185); GAF, GAF domain (218 to 402); PHY, PHY domain (413 to 593). The four small rectangles in the figure indicate the chromophores. The most and second-most important components of each phyA-specific function are indicated by black and gray lines, respectively.