Article Figures & Data
Figures
- Fig. 1.
Phenotype characterization of the dominantphyA-300D mutant. A, Three-day-old FRc-grownphyA-300D homozygote (−/−), heterozygote (−/+), and wild-type (WT) seedlings. Average hypocotyl lengths (in millimeters) are shown above each seedling (n = 25) withsd shown in parentheses. B, Hypocotyl length of wild-type (WT) and phyA-300D seedlings grown in continuous white (W) and blue (B) light and Rc (R) and FRc (FR), or in darkness (D) for 6 d. Each column represents a mean value of the hypocotyl length (in millimeters) taken from 25 seedlings with thesd indicated by an error bar.
- Fig. 2.
The structural features of phyA and the molecular lesion of the phyA-300D mutation. A, Diagram of the Arabidopsis PHYA protein. The chromophore-binding site (C-323), core region, and its two PAS motifs are indicated. B, The conserved Val and its surrounding amino acid residues among four Arabidopsis phytochromes. Note that the Val is invariant among all known phyA species from higher plants, although it can be substituted by Ala in all other phytochromes among different higher plant species. The single-letter codes of amino acids are shown in bold. C, Nucleotide sequence comparison of the PHYA gene in phyA-205,phyA-300D, and wild type (WT) around the Val-631 region. Amino acids (in bold single-letter code) are shown above their codons. In both mutant alleles, there is a single identical amino acid residue mutation (V631M) in the translated proteins.
- Fig. 3.
The phyA-205 has a dominant-negative phenotype and is not dependent on Suc concentration. Average hypocotyl lengths (in millimeters) are shown above each seedling (n = 25) with the sd shown in parentheses. A, Phenotypic comparison of 3-d-old FR light-grownphyA-205 homozygous (−/−), heterozygous (−/+), and wild-type (WT) seedlings on 0.3% (w/v) Suc medium. B, Phenotypic comparison of 3-d-old FR light-grown phyA-205homozygous (−/−), heterozygous (−/+), and wild-type (WT) seedlings on 2% (w/v) Suc medium.
- Fig. 4.
Expression of PHYA-V631M mutant protein is sufficient to cause a dominant interfering effect on endogenous phyA signaling. A, Diagrams of the transformation cassettes. The wild-typePHYA (WT) and PHYA-V631M full-length coding regions were driven by the cauliflower mosaic virus 35S promoter. B, Morphological comparison of 3-d-old FR light-grown seedlings of wild-type PHYA overexpressor (PHYA OE), wild-type (WT), and two mutant PHYA-V631M overexpressor (PHYA-300D OE) lines. PHYA protein immunoblots are shown in the bottom panel. C, Morphological comparison of 3-d-old dark-grown seedlings with PHYA-V631M overexpressor transgene in wild-type background (left) and the wild-type control (right). D, Morphology of 3-d-old FR light-grown seedling with PHYA-V631M overexpressor transgene in a phyA null mutant background. Scale bars in B through D = 1 mm.
- Fig. 5.
Characterization of wild-type and PHYA-V631M stability under R and FR light. A, phyA levels in 3-d-old dark (D) and FR light-grown wild-type Columbia (WT) and phyA-300D, and in 3-d-old dark-grown seedlings exposed to Rc for 1 or 4 h (hr; top). The immunoblots were probed with a phyA-specific polyclonal antibody. An equal amount of total protein was loaded in each lane, as verified by CSN5 immunoblot (bottom). B, phyA levels in 3-d-old dark-grown (D) wild-type Columbia (WT), and phyA-300D seedlings and in 3-d-old dark-grown seedlings exposed to FRc light for 1, 4, or 24 h (hr). An equal amount of total protein was loaded in each lane, as verified by immunoblot using anti-tubulin antibodies (bottom).
- Fig. 6.
Native gel analysis of phyA dimerization in FR and dark-grown wild-type and mutant seedlings. Extracts were separated on a 4% to 20% (w/v) gradient non-denaturing gel and probed with a polyclonal PHYA antibody. The phyA dimer is marked by a triangle on the right. Equal loading was ensured by visualizing a protein band staining intensity (marked by control). The positions of two mass markers are indicated on the left side. The phyA-101 is a null mutant control. A, FR light-grown seedlings (5 d); B, dark-grown seedlings (5 d).
- Fig. 7.
The PHYB-V664M protein is physiologically active and does not confer an interfering effect on phyB signaling. The 3-d-old seedlings are shown on top, and their corresponding PHYB protein immunoblots are shown on the bottom. A, Three-day-old Rc light-grown wild type (WT), PHYB overexpressor (PHYB OE) in wild-type background, and the phyB-101 null mutant. B, Three-day-old Rc light-grown seedlings of three independentPHYB-V664M overexpressor lines (L1, L2, and L3 ofPHYB-V644M OE) in the phyB-101 null mutant background. C, Three-day-old Rc light-grown seedlings of two differentPHYB V644M overexpressor (PHYB-V644M OE) lines in a wild-type background. L′1 is an overexpressor line for PHYB-V664M, and L′2 is a cosuppression line.
Tables
Isolate Name Allele Nature of Mutation 128 fhy3-128 Recessive 6476 fhy3-6476 Recessive A2GO5 phyA-300D Dominant 135 phyA-301 Recessive 158 hy5-158 Recessive CS19960 fhy1-19960 Recessive 149 fhy1-149 Recessive A2GO6 fhy4-1 Recessive Cross Generation Seedlings with Short Hypocotyl Seedlings with Long Hypocotyl phyA-300D × WT F1 0 69 F2 188 677 phyA-300D ×phyA-1 F1 0 2 F2 0 400 phyA-205 × WT F1 0 14 F2 105 370 phyA-205 ×phyA-300D F1 0 9 F2 0 200 phyA-301 × WT F1 30 0 F1 and F2 progeny of the complementation crosses were grown in continuous far red light for 6 d before scoring seedling phenotypes and their segregation.
