Blue light-induced autophosphorylation of phototropin is a primary step for signaling

Abstract

Phototropins are autophosphorylating protein kinases of plant-specific blue light receptors. They regulate various blue light responses, including phototropism, chloroplast movements, hypocotyl growth inhibition, leaf flattening, and stomatal opening. However, the physiological role of autophosphorylation remains unknown. Here, we identified phosphorylation sites of Ser or Thr in the N terminus, Hinge1 region, kinase domain, and C terminus in Arabidopsis phototropin1 (phot1) by liquid chromatography-tandem mass spectrometry in vivo. We substituted these Ser or Thr residues with Ala in phot1 and analyzed their functions by inspecting the phot1-mediated responses of stomatal opening, phototropism, chloroplast accumulation, and leaf flattening after the transformation of the phot1 phot2 double mutant. Among these sites, we found that autophosphorylation of Ser-851 in the activation loop of the kinase domain was required for the responses mentioned above, whereas the phosphorylation of the other Ser and Thr, except those in the activation loop, was not. Ser-849 in the loop may have an additional role in the responses. Immunological analysis revealed that Ser-851 was phosphorylated rapidly by blue light in a fluence-dependent manner and dephosphorylated gradually upon darkness. We conclude that autophosphorylation of Ser-851 is a primary step that mediates signaling between photochemical reaction and physiological events.

Fig. 1.

Identification of the phosphorylation sites of Arabidopsis phot1. (A) Blue light-induced autophosphorylation of phot1 in vivo in Arabidopsis. Etiolated seedlings labeled by ³²P were kept in the dark (Dk) or illuminated with blue light at 100 μmol m⁻²·s⁻¹ for 1 min (BL). The phot1 protein was isolated by immunoprecipitation from the microsomes of the seedlings, and separated by SDS/PAGE. An autoradiogram is illustrated. (B) Phosphorylation-dependent binding of a 14-3-3 protein to the immunopurified phot1. Protein blot of phot1 was done with glutathione-S-transferase (GST)-GF14φ. (C) Coomassie brilliant blue stain of immunopurified phot1. (D) A typical case of a phosphorylation site mapping in phot1 by LC-MS/MS. Phot1 protein was digested with trypsin in gel. A MS/MS spectrum of a phosphorylated peptide ⁴⁰⁸KSpSLSFMGIK⁴¹⁷ is represented. pS indicates phosphorylated Ser. (E) All identified phosphorylation sites in phot1. Individual phosphopeptide sequences and positions of Ser and Thr are indicated. Asterisks indicate the peptides identified in error-tolerant search against phot1. Double asterisks indicate that a precise phosphorylation site was not determined because of the lack of informative fragment ions.

Fig. 2.

Autophosphorylation of the Ser-851 in the activation loop of phot1 in vivo by blue light. (A) The phosphorylation of Ser-851. Phot1 was immunopurified from 200 μg of proteins of microsomal membranes as shown in Fig. 1. The obtained phot1 was visualized with anti-pSer-851 antibodies (Upper) and immunodetected (Lower). (B) Fluence dependencies in phosphorylation of the Ser-851 in response to blue light. Etiolated seedlings were irradiated by a pulse of blue light for 30 sec at the indicated fluence rates. The seedlings were disrupted 1 min after the start of blue light, and microsomal membranes were immediately isolated from the seedlings. The membranes of 40 and 20 μg of protein were used for the immunoblots of pSer-851 (Upper) and phot1 (Lower), respectively. (C) Time courses of phosphorylation and dephosphorylation of the Ser-851 in response to blue light. The seedlings were irradiated by blue light at 100 μmol m⁻²·s⁻¹ for 30 sec and were disrupted at indicated times after the start of blue light. Microsomal membranes were immediately isolated, and used for immunoblots of pSer-851 (Upper) and phot1 (Lower). Experiments repeated on three occasions gave similar results.

Fig. 3.

Blue light-induced stomatal responses in transgenic plants. (A) Stomatal opening in the epidermis. Epidermal peels were irradiated by red light (60 μmol m⁻²·s⁻¹; RL) or red (50 μmol m⁻²·s⁻¹) and blue (10 μmol m⁻²·s⁻¹; RL + BL) light for 3 h. Values are means of three independent experiments with standard deviations, with 45 stomata measured in each experiment. (B) Blue light-dependent H⁺ pumping in guard cell protoplasts, determined by pH decrease. Guard cell protoplasts (100 μg of proteins) were irradiated with red light at 600 μmol m⁻²·s⁻¹ and superimposed with blue light at 100 μmol m⁻²·s⁻¹ for 30 sec. The protoplasts were added by 10 μM fusicoccin. Measurements were done at 24°C. BL, blue light; FC, fusicoccin. Experiments repeated on three occasions gave similar results. (C) Expression of the phot1 proteins in the transgenic Arabidopsis plants. Immunoblot of the phot1 protein in gl1, the phot1 phot2 double mutant, and all of the transgenic plants. Immunoblot was performed by using 20 μg of microsomal proteins prepared from etiolated seedlings. Experiments repeated on two occasions gave similar results.

Fig. 4.

Phot1-mediated responses in transgenic plants. (A) Phototropism. Etiolated seedlings were irradiated with unilateral blue light at 0.5 μmol m⁻²·s⁻¹ for 14 h. Values are means of 49–70 hypocotyls with standard errors. (B) Slit assays of chloroplast accumulation. Detached leaves were irradiated with blue light at 2.5 μmol m⁻²·s⁻¹ for 30 min through a slit of 1-mm width. Arrowheads indicate the irradiated areas. Experiments repeated on two occasions gave similar results. (C) Leaf flattening. The plants were grown under white light at 50 μmol m⁻²·s⁻¹ for 3 weeks. (Scale bar, 1 cm.) Experiments repeated on two occasions gave similar results.

Fig. 5.

Autophosphorylation kinase activity in vivo. Autophosphorylation activity by phot1 kinase in vivo was determined through protein blot (A), staining of phosphorylation (B), and mobility shift (B). Etiolated seedlings were kept in the dark (Dk) or irradiated with a blue light pulse at 100 μmol m⁻²·s⁻¹ for 1 min (BL), and microsomal membranes were immediately isolated from the seedlings. (A) Phot1 in the membranes was separated by SDS/PAGE and transferred to nitrocellulose membrane. The nitrocellulose membrane was incubated with GST-GF14φ to measure the phosphorylation-dependent binding of a 14-3-3 protein to phot1 (Upper). Immunoblot of phot1 (Lower). (B) Phot1 was immunoprecipitated from 200 μg of the microsomal membranes. The immunopurified phot1 proteins were separated by SDS/PAGE and stained by phos-tag (Upper). Immunoblot of phot1 (Lower). Experiments repeated on three occasions gave similar results.