Cryptochrome-mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis

Abstract

Both phototropins (phot1 and phot2) and cryptochromes (cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids (GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high-intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3 (nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose-containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3. Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.

Figure 1

Functions of cryptochromes and phototropins in hypocotyl curvature induced by different intensities of blue light in the presence or absence of sucrose (A, C) Hypocotyl phototropism in 3‐d‐old etiolated seedlings of the wild type (WT) and phot1 phot2, cry1 cry2, and phot1 phot2 cry1 cry2 Arabidopsis mutants grown on the same vertical plates and exposed to blue light (BL) illumination at different intensities (from 0.01 to 100 μmol · m⁻² s⁻¹) for 12 h in medium with sucrose (A) or without sucrose (C). (B) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings of A. The values are the means ± SD of three independent experiments (28–31 seedlings each). (D) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings of (C). The values are the means ± SD of three independent experiments (34–36 seedlings each). Scale bar = 5 mm.

Figure 2

Effects of sucrose on hypocotyl curvature in wild‐type and different mutant seedlings (A, C) Hypocotyl phototropism of 3‐d‐old etiolated seedlings of the wild type (WT) and rpt2‐2, nph3‐6, phot1 phot2, and pks1 pks2 pks4 Arabidopsis mutants grown on the same vertical plates and exposed to BL illumination at different intensities (from 0.01 to 100 μmol · m⁻² s⁻¹) for 12 h in medium with sucrose (A) or without sucrose (C). (B) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (A). The values are the means ± SD of three independent experiments (25–28 seedlings each). (D) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (C). The values are the means ± SD of three independent experiments (32–35 seedlings each). Scale bar = 5 mm.

Figure 3

Effects of sucrose and GA₃ on hypocotyl phototropism in wild‐type seedlings and the blue light photoreceptor mutants (A, C) Hypocotyl phototropism in 3‐d‐old etiolated seedlings of the wild type (WT) and phot1 phot2, cry1 cry2, and phot1 phot2 cry1 cry2 Arabidopsis mutants grown on the same vertical plates and exposed to BL illumination at a fluence rate of 0.01 or 100 μmol · m⁻² s⁻¹ for 12 h in medium with sucrose (MS), without sucrose (MS‐S), or MS plus 1 (MS + 1 μM GA₃) or 10 μmol · L⁻¹ GA₃ (MS + 10 μM GA₃). (B) Hypocotyl curvature was measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (A). The values are the means ± SD of three independent experiments (31–33 seedlings each). (D) Hypocotyl curvature was measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (C). The values are the means ± SD of three independent experiments (29–32 seedlings each). Scale bar = 5 mm.

Figure 4

Effects of sucrose and GA on hypocotyl phototropism mediated by NPH3 and RPT2 (A) Hypocotyl phototropism of 3‐d‐old etiolated seedlings of the wild type (WT) and nph3‐6, phot1 nph3‐6, and phot2 nph3‐6 Arabidopsis mutants grown on the same vertical plates and exposed to BL illumination at a fluence rate of 100 μmol · m⁻² s⁻¹ for 12 h in the presence of sucrose (MS), without sucrose (MS‐S), or MS plus GA₃ (MS +GA₃). (B) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (A). (C) Hypocotyl curvature of 3‐d‐old etiolated seedlings of the wild type (WT) and rpt2‐2, phot1 rpt2‐2, phot2 rpt2‐2, and phot1 phot2 Arabidopsis mutants grown on the same vertical plates and exposed to BL illumination at a fluence rate of 100 μmol · m⁻² s⁻¹ for 12 h in the presence of sucrose (MS), without sucrose (MS‐S), or MS plus GA₃ (MS+GA₃). (D) Hypocotyl curvature measured as the change in hypocotyl angle determined from an analysis of etiolated seedlings in (C). Scale bar = 5 mm.

Figure 5

Auxin distribution of wild type in response to GA₃ or Suc and the expression of DELLAs in the hypocotyls (A) GUS staining patterns in the DR5:GUS wild‐type (Col) seedlings. Three‐d‐old WT seedlings harboring DR5:GUS. From left to right: etiolated dark‐grown seedling (Dark), seedling grown in continuous 100 µmol · m⁻²s⁻¹ blue light (HBL), HBL‐grown seedling in MS or MS‐S, HBL‐grown seedling with 10 µmol · L⁻¹ GA₃ (HBL+GA₃). Arrowheads indicate the asymmetric expression of GUS in the hypocotyls. Scale bars = 5 mm. (B) RGA stability after addition of 90 mmol/L sucrose (MS+S) or without sucrose (MS‐S) in the WT and phot1 phot2, cry1 cry2, and phot1 phot2 cry1 cry2 mutant seedlings irradiated by high‐intensity blue light for 3 h. Ponceau staining and specific bands serve as equal loading controls. Scale bar = 1 mm.

Figure 6

GA and IAA contents in etiolated seedlings exposed to blue light in sucrose‐free and sucrose‐containing medium (A) GA₄ content in 3‐d‐old etiolated seedlings exposed to 100 µmol · m⁻²s⁻¹ of blue light for 6 h or no in sucrose‐free medium and 3% sucrose‐containing medium. (B) IAA content in 3‐d‐old etiolated seedlings of different mutants exposed to100 µmol · m⁻²s⁻¹ of blue light for 6 h in sucrose‐free medium and 3% sucrose‐containing medium. (C) GA₈, GA₁₂, GA₁₉, GA₂₄ and GA₃₄ content in 3‐d‐old etiolated seedlings exposed to 100 µmol · m⁻²s⁻¹ of blue light for 6 h in sucrose‐free medium and 3% sucrose‐containing medium. Values are the means ± SD of three replicates. phot1 phot2 (P1P2), cry1 cry2 (C1C2), and phot1 phot2 cry1 cry2 (P1P2C1C2).

Figure 7

RT‐qPCR analysis of the expression of PRE6, SAUR9, IAA19, RPT2, ABCB19, and PIF4 in the hypocotyl of etiolated Arabidopsis seedlings under the different treatments (A–F) RT‐qPCR analysis of the expression of PRE6, SAUR9, IAA19, RPT2, ABCB19, and PIF4 in the hypocotyl of the WT and phot1 phot2, cry1 cry2, and phot1 phot2 cry1 cry2 mutant seedlings irradiated by high‐intensity blue light for 1 h or 3 h in the presence of sucrose (MS), without sucrose (MS‐S), and MS plus GA₃ (MS+GA₃). The values are the average of three independent experiments with SD.

Figure 8

Model for phototropic adaptation mechanisms Continuous irradiation with high‐intensity blue light activates PHOT1, which interacts with NPH3‐RPT2 complexes, whereas PHOT2 regulates the reconstruction of the phot1‐NPH3 complex for its re‐localization back to the plasma membrane. The phot signal is transduced through the RPT2‐NPH3 complexes to induce the asymmetric distribution of auxin and asymmetric hypocotyl growth. On the other hand, upon HBL irradiation, cryptochromes inhibit the GA synthesis to stabilize DELLAs to control the PIFs transcription module in the regulation of hypocotyl growth. By contrast, sucrose blocks the GA‐mediated degradation of DELLAs to inhibit hypocotyl growth. So, it is possible that the release of hypocotyl growth inhibited via reducing the DELLA protein abundance by GAs or depletion of sucrose, supporting the bending growth.