Development and anthocyanin content of wild-type, transgenic BVR and phy mutant seedlings under continuous blue light. No-0 WT, 35S::pBVR3, CAB3::pBVR2 (CAB-2), Col-0 WT, phyA (Salk_014575) and phyB (Salk_022035) seedlings were grown at 22°C on Phytoblend medium with or without 1% sucrose for ∼4 days under Bc illumination of 25 µmol m⁻²s⁻¹. (A) Image of seedlings grown on 1% sucrose; arrows indicate visible anthocyanin. Scale bar represents 1 cm. (B) Image of seedlings grown in the absence of sucrose, Scale bar represents 1 cm. (C) Anthocyanin content. Black bars (1% sucrose) and white bars (no sucrose), represent the mean (±SD) of three independent measurements. Fold-increase values for anthocyanin levels determined for seedlings grown on 1% sucrose relative to 0% sucrose are indicated.

Development and anthocyanin content of wild-type and phy mutant seedlings under continuous red light. Col-0 WT, phyA (Salk_014575), phyB (Salk_022035), phyC (Salk_007004), phyD (Salk_027336) and phyE (Salk_092529) seedlings were grown at 22°C on Phytoblend medium with or without 1% sucrose for ∼4 days under Rc illumination of 50 µmol m⁻²s⁻¹. (A) Image of seedlings grown on 1% sucrose; arrow indicates visible anthocyanin. Scale bar represents 1 cm. (B) Image of seedlings grown in the absence of sucrose. Scale bar represents 1 cm. (C) Anthocyanin content. Black bars (1% sucrose) and white bars (no sucrose), represent the mean (±SD) of at least three independent measurements. Fold-increase values for anthocyanin levels determined for seedlings grown on 1% sucrose relative to 0% sucrose are indicated.

Model of the roles of phytochromes in the photoregulation of anthocyanin content and hypocotyl elongation. (A)Role for mesophyll-localized phytochromes in the photoregulation of anthocyanin and hypocotyl elongation under continuous blue (Bc), far-red (FRc) and red (Rc) illumination based on analyses of transgenic CAB3::pBVR lines. (B) Roles of individual phytochrome family members in the Rc-dependent photoregulation of anthocyanin based on analyses of individual phy mutant lines.

Analyses of T-DNA insertions and phytochrome transcript accumulation in apophytochrome T-DNA mutants. (A) Sites of T-DNA insertions in phyA (SALK_014575), phyB (SALK_022035), phyC (SALK_007004), phyD (SALK_027336) and phyE (SALK_092529) mutants are shown. Arrowheads indicate annealing locations of gene-specific oligonucleotides used in RT-PCR analysis. FP, forward primer and RP, reverse primer. (B) RT-PCR analyses of the transcript accumulation of PHYA, PHYB, PHYC, PHYD and PHYE in T-DNA insertion apophytochrome mutants was performed using primers that span or are downstream of the T-DNA insertion site in the respective genes to analyze transcript accumulation. RNA was extracted from Columbia-0 (Col-0) wild-type, phyA (SALK_014575), phyB (SALK_022035), phyC (SALK_007004), phyD (SALK_027336) and phyE (SALK_092529) seedlings grown at 22°C on Phytoblend medium containing 1% Suc for 4 d under Rc illumination of 50 µmol m⁻²s⁻¹. The expression of UBC21 (At5g25760) transcript was analyzed as an internal control. Numbers to left indicate sizes of standard DNA fragments in base pairs.

Sucrose-dependent anthocyanin accumulation in wild-type, phyE mutant and complemented P_PHYE:phyE-myc₆/phyE mutants. (A) Columbia-0 wild-type (Col-0 WT), two independent complemented phyE mutants (phyE-myc6-1 and phyE-myc6-11) and phyE were grown at 22°C on Phytoblend medium with or without 1% sucrose for four days under Rc illumination of 50 µmol m⁻²s⁻¹. Black bars (1% sucrose) and white bars (no sucrose), represent the mean (±SD) of three independent measurements. Fold-increase values for anthocyanin levels determined for seedlings grown on 1% sucrose relative to 0% sucrose are indicated. (B) phyE null mutant was complemented with a transgene driven by the PHYE native promoter encoding a C-terminal myc epitope-tagged phyE (pBI-P_PHYE:phyE-myc₆). For immunoblot analysis of phyE accumulation in two independent complemented phyE mutants (phyE-myc6-1 and phyE-myc6-11), transgenic lines were grown at 22°C on Phytoblend medium with 1% sucrose for 10 days under Rc illumination of 50 µmol m⁻²s⁻¹. Soluble protein extracts (∼60 µg) were used for immunoblot analysis with anti-myc antibodies. Numbers to right indicate molecular mass in kilodaltons. The band above 120 kD represents the phyE-myc protein, whereas the band near 80 kD is a non-specific band.