Phytochrome A Mediates Blue-Light Enhancement of Second-Positive Phototropism in Arabidopsis

Abstract

Hypocotyl phototropism of etiolated Arabidopsis seedlings is primarily mediated by the blue-light receptor kinase phototropin 1 (phot1). Phot1-mediated curvature to continuous unilateral blue light irradiation (0.5 μmol m(-2) s(-1)) is enhanced by overhead pre-treatment with red light (20 μmol m(-2) s(-1) for 15 min) through the action of phytochrome (phyA). Here, we show that pre-treatment with blue light is equally as effective in eliciting phototropic enhancement and is dependent on phyA. Although blue light pre-treatment was sufficient to activate early phot1 signaling events, phot1 autophosphorylation in vivo was not found to be saturated, as assessed by subsequently measuring phot1 kinase activity in vitro. However, enhancement effects by red and blue light pre-treatment were not observed at higher intensities of phototropic stimulation (10 μmol m(-2) s(-1)). Phototropic enhancement by red and blue light pre-treatments to 0.5 μmol m(-2) s(-1) unilateral blue light irradiation was also lacking in transgenic Arabidopsis where PHOT1 expression was restricted to the epidermis. Together, these findings indicate that phyA-mediated effects on phot1 signaling are restricted to low intensities of phototropic stimulation and originate from tissues other than the epidermis.

Keywords: blue light; epidermis; phosphorylation; phototropin; phototropism; phytochrome; red light.

Figure 1

Pre-irradiation with red light enhances phototropic responsiveness of etiolated seedlings in a phyA-dependent manner. Three-day-old etiolated WT (A) and phyA (B), phyB (C) and phyA phyB (D) mutant seedlings were maintained in darkness (Control) or irradiated with 20 μmol m⁻² s⁻¹ of over-head red light for 15 min (Red) before being placed into 0.5 μmol m⁻² s⁻¹ of unilateral blue light for 3 h. Hypocotyl curvatures were measured every 10 min and each value is the mean ± S.E. of 18–20 seedlings. Asterisks indicate significant differences between red light treated and control seedlings (P < 0.001, Student's t-test).

Figure 2

Pre-irradiation with blue light enhances phototropic responsiveness of etiolated seedlings in a phyA-dependent manner. Three-day-old etiolated WT (A) and phyA (B), phyB (C) and phyA phyB (D) mutant seedlings were maintained in darkness (Control) or irradiated with 20 μmol m⁻² s⁻¹ of over-head blue light for 15 min (Blue) before being placed into 0.5 μmol m⁻² s⁻¹ of unilateral blue light for 3 h. Hypocotyl curvatures were measured every 10 min and each value is the mean ± S.E. of 17–20 seedlings. Control data as in Figure 1. Asterisks indicate significant differences between blue light treated and control seedlings (P < 0.001, Student's t-test).

Figure 3

Pre-irradiation does not enhance phototropic responsiveness to high intensity unilateral blue light. (A) Three-day-old etiolated WT seedlings were maintained in darkness (Control) or irradiated with 20 μmol m⁻² s⁻¹ of over-head red light for 15 min (Red) or (B) irradiated with 20 μmol m⁻² s⁻¹ of over-head blue light for 15 min (Blue) before being placed into 10 μmol m⁻² s⁻¹ of unilateral blue light for 3 h. Hypocotyl curvatures were measured every 10 min and each value is the mean ± S.E. of 20 seedlings.

Figure 4

Blue-light mediated changes in phot1 and NPH3 phosphorylation status. Immunoblot analysis of total protein extracts from 3-day-old etiolated WT and phot1 phot2 (p1p2) mutant seedlings maintained in darkness (D) or irradiated with 20 μmol m⁻² s⁻¹ of over-head blue light for 15 min (L). Protein extracts were probed with anti-phot1 (upper panel), anti-NPH3 antibodies (middle panel) and anti-UGPase antibody as a loading control (lower panel). The dashed lines indicate the highest mobility edge.

Figure 5

Effect of in vivo blue light irradiation on in vitro light-dependent phot1 kinase activity. Total protein extracts were made from 3-day-old etiolated seedlings maintained in darkness (D) or given one of three different in vivo blue-light treatments: 0.5 μmol m⁻² s⁻¹ for 30 min (Low Blue), 20 μmol m⁻² s⁻¹ for 15 min (High Blue), or 20 μmol m⁻² s⁻¹ for 15 min followed by 0.5 μmol m⁻² s⁻¹ for 30 min (High Blue + Low Blue). (A) Autoradiograph of in vitro kinase assays of total protein extracts given an in vitro mock irradiation (D) or irradiated with white light (L) at a total fluence of 30,000 μmol m⁻² upon addition of radiolabeled ATP. Immunoblot analysis of protein levels with anti-phot1 antibody and anti-UGPase antibody as a loading control is shown in the lower panel. (B) Quantification of in vitro light-dependent phot1 kinase activity in total protein extracts. Kinase activity was quantified and expressed as a percentage of maximal autophosphorylation.

Figure 6

Epidermal-specific expression of phot1-GFP in ML1::PHOT1-GFP transgenic lines. (A) Phot1-GFP localization in hypocotyls of 3-day-old etiolated seedlings. Reconstructed hypocotyl cross sections of PHOT1::PHOT1-GFP (P1::P1-GFP) and ML1::PHOT1-GFP (ML1::P1-GFP) expressing seedlings. FM4-64 staining was used to define cell layers. Ep, epidermis; C1 and C2, cortex layers 1 and 2, respectively; En, endodermis. Scale bar, 50 μm. (B) Slit band assays of chloroplast accumulation in wild type (WT), phot1 phot2 mutants (p1p2) and plants expressing P1::P1-GFP and ML1::P1-GFP. Detached leaves were irradiation with 1.5 μmol m⁻² s⁻¹ of blue light through a 1 mm slit for 60 min. Arrows indicate irradiated areas.

Figure 7

Pre-irradiation does not enhance phototropic responsiveness in ML1::PHOT1-GFP transgenic lines. Three-day-old etiolated seedlings expressing ML1::PHOT1-GFP (A) or PHOT1::PHOT1-GFP (B) were maintained in darkness (Control), irradiated with 20 μmol m⁻² s⁻¹ of over-head red light for 15 min (Red) or irradiated with 20 μmol m⁻² s⁻¹ of over-head blue light for 15 min (Blue) before being placed into 0.5 μmol m⁻² s⁻¹ of unilateral blue light for 3 h. Hypocotyl curvatures were measured every 10 min and each value is the mean ± S.E. of 16–21 seedlings. Asterisks indicate significant differences between red or blue light treated and control seedlings (P < 0.001, Student's t-test).