Figure 1.
phyB and phyC proteins form complexes in both etiolated and light-grown rice seedlings.
A. Protein extracts (200 µg) from 7-day-old etiolated seedlings of WT or phyC mutants (phyC) were immunoprecipitated with an anti-PHYC antibody (Anti-PHYC/WT; Anti-PHYC/phyC) or with preimmune serum from the same rabbit (Pre/WT). phyC or phyB was immunodetected in the precipitation. Thirty micrograms of protein extracts from WT were loaded as the positive control (Ext). B. Protein extracts (500 µg) from WT seedlings grown under W for 7 days were immunoprecipitated with an anti-PHYC antibody (Anti-PHYC/WT) or with preimmune serum from the same rabbit (Pre/WT). phyC or phyB was immunodetected in the precipitation. Fifty micrograms of protein extracts from WT were loaded as the positive control (Ext).
Figure 2.
Immunoblot analyses of phyA, phyB, and phyC in the column fractions of protein extracts.
Protein was extracted from 7-day-old etiolated seedlings of WT and all phytochrome single and double mutants and fractionated by SEC. phyA, phyB, and phyC were immunochemically detected with anti-PHYA (A), anti-PHYB (B), and anti-PHYC antibodies (C), respectively, in the individual fractions (#17–#24). Small numbers above the fraction numbers are the molecular sizes which were calculated based on the calibration line of standard proteins. Arabidopsis seedlings were used to characterize the migration of homodimer phyA. D. Physical interactions between phyB and phyC in the fractions (#17–#26) of protein extracts from 7-day-old etiolated seedlings of WT. The individual fractions were immunoprecipitated with an anti-PHYC antibody. phyB and phyC in the precipitates were detected by immunoblotting. Thirty micrograms of protein extracts from WT were loaded as the positive control (Ext).
Figure 3.
phyC-GFP is biologically active in phyA phyC backgrounds but inactive in phyB-mutant backgrounds.
A. Diagram of the PHYC-GFP fusion construct introduced into phyA phyC and phyB mutants. B. Immunoblot analyses of phyB and the phyC-GFP fusion protein in WT, phyA phyC, and PCG/aacc transgenic lines using anti-PHYB and anti-PHYC antibodies. Protein extracts (25 µg from PCG/aacc lines and PCG/Aabb lines; 50 µg from WT and phyA phyC seedlings) were loaded to detect phyC-GFP and phyC. Fifty micrograms of protein were loaded to detect phyB. Aabb is the phyB mutant where the PHYA mutant allele is heterozygous. C. The phyC-GFP fusion protein caused inhibition of coleoptile growth by FR irradiation in PCG/aacc transgenic lines. Mean coleoptile lengths are shown for WT, phyA, phyA phyC, and PCG/aacc seedlings (#19 and #20) grown under FR (15 µmol m−2 s−1, open bars) or in the dark (filled bars) for 8 days. The mean ± SE (standard error) obtained from at least 12 seedlings is plotted. D. phyC-GFP fusion protein exerted the phyC function in the expression of RbcS. The expression of RbcS induced by FR was comparatively analyzed by RNA blotting in the seedlings of WT, phyA, phyA phyC, and PCG/aacc (#19 and #20). RbcS was used as a probe. rRNA was stained by methylene blue as a quantity control. E. phyC-GFP did not participate in the photoinhibitory responses of coleoptile growth under FR or R in the phyB mutant background. Mean coleoptile lengths are shown for WT, phyB, phyA phyB, PCG/Aabb, and PCG/aabb seedlings grown under FR (15 µmol m−2 s−1, open bars) or under R (15 µmol m−2 s−1, hatched bars) or in the dark (filled bars). The mean ± SE obtained from at least 12 seedlings is plotted, excluding the PCG/aabb genotype, for which only six seedlings were grown.
Figure 4.
Absorption difference spectra of phyC-GFP.
Difference spectrum characteristics of phytochromes in protein extracts from 7-day-old etiolated seedlings of PCG/aabb (A) or PCG/Aabb (B). Difference spectra were measured using a double-beam spectrophotometer and normalized by 1 mg total protein per mL. C. Immunoblot analyses of phyA and the phyC-GFP fusion protein in PCG/Aabb or PCG/aabb transgenic lines using anti-PHYA and anti-PHYC antibodies. Each lane was loaded with 5 µg of total protein.
Figure 5.
phyC is biologically active in PHYB/aabb and PHYB(C364A)/aabb transgenic lines.
A. Diagram of PHYB and mutant PHYB(C364A) constructs introduced into phyB mutants. B. Immunoblot analyses of phyB and phyC levels in the dark-grown seedlings of PHYB/Aabb and PHYB(C364A)/Aabb transgenic lines. Protein extracts from 7-day-old etiolated seedlings of different transgenic lines of PHYB(C364A)/Aabb (#27, #69, #71, and #72) and PHYB/aabb (#7 and #11) were probed with anti-PHYC and anti-PHYB antibodies. WT and phyB were used as positive and negative controls, respectively. Each lane was loaded with 50 µg of protein extracts. C. FR inhibited coleoptile growth in PHYB/aabb transgenic lines. Mean coleoptile lengths were shown for the seedlings of WT, phyA, phyB, phyA phyB, and two lines of PHYB/aabb (#7-2- and #11-3-) grown under FR (15 µmol m−2 s−1, open bars) or in the dark (filled bars) for eight days. The mean ± SE obtained from at least 12 seedlings is plotted. D and E. FR (D) or R (E) inhibited coleoptile growth in the PHYB(C364A)/Aabb and PHYB(C364A)/aabb transgenic lines. Mean coleoptile lengths are shown for WT, phyA (only in D), phyB, phyA phyB, PHYB(C364A)/Aabb, and PHYB(C364A)/aabb seedlings grown under FR (15 µmol m−2 s−1, D), R (15 µmol m−2 s−1, E), or in the dark (filled bars) for eight days. Four independent lines (#27-6-, #69-2-, #71-1-, and #72-1-) of PHYB(C364A) were used, and results from different genetic backgrounds (Aabb or aabb) were depicted with separate bars. The mean ± SE obtained from at least 12 seedlings is plotted.
Figure 6.
PHYB(C364A)/Aabb transgenic lines flower earlier than WT under LD conditions.
Nipponbare (WT), phyB-1 mutant (phyB), and PHYB(C364A)/Aabb transgenic lines were grown in a growth chamber set with LD (14.5L/9.5D) condition. The mean ± SE obtained from 20 plants is shown.
Figure 7.
phyB is involved in light-induced degradation of phyC in rice.
A. Levels of phyC are not reduced in phyB mutant seedlings grown under W. Protein extracts were prepared from WT and phyB seedlings grown under W for 7 days. Each lane was loaded with 50 µg of protein extracts for the detection of phyB and phyC using anti-PHYB and anti-PHYC antibodies, respectively. Nipponbare (Nip) and Norin8 (N8) were used as controls. phyB-1, -2, -3, -4, and -5 are five different mutant alleles of PHYB. B. Effect of W on the transcript levels of PHYA and PHYC genes in WT and the phyB mutant. The seedlings of WT and phyB-1 mutant (phyB) were grown for 5 days in the dark (D) and then exposed to W for 4, 8, 12, or 24 h before harvesting. For detecting the transcripts of PHYA and PHYC, each lane was loaded with 10 µg of total RNA. As a quantity control, rRNA was stained with methylene blue. C. Effect of W on phyA and phyC protein concentrations in WT and the phyB mutant. Growth conditions of the seedlings were the same as those in (B). Fifty micrograms of protein extract were loaded in each lane. D. PHYB(C364A) protein is necessary for the R-induced degradation of phyC. PHYB(C364A)/aabb (#27) and PHYB/aabb (#11) seedlings were grown in the dark (D) or in the D and then exposed to R for 8 or 24 h before harvesting. phyB and phyC proteins in 50 µg of protein extracts were detected with anti-PHYB and anti-PHYC antibodies, respectively.
Figure 8.
State models of phyC and phyB in rice seedlings.
A. In the cells of WT seedlings grown in the dark, most of the phyC exists as phyB/phyC heterodimers (B/C) and, probably, to a smaller degree as phyC monomers (C?), while phyB exist as phyB/phyC heterodimers (B/C) and phyB/phyB homodimers (B/B). In etiolated seedlings, phyB stabilizes phyC in the B/C conformation. Consequently, phyC levels are quite low in the phyB-deficient mutants. B. When seedlings are exposed to light, including W, R, and FR, phyC subunits in phyB/phyC heterodimers (B/C) are light-labile and biologically active to participate in the multiple processes of rice development (inhibition of coleoptile growth, induction of light-regulated genes, and chlorophyll accumulation). By contrast, phyC monomers (C) are light-stable but do not participate in the de-etiolation of rice seedlings. phyB in its heterodimeric (B/C) and homodimeric (B/B) forms probably play different roles in the responses to light.