Fig 1.
Diagram showing the transitional light intensity for the accumulation and avoidance responses.
Solid vertical lines indicate points of the transitional light intensity under warm (A; 22°C) and cold (B; 5°C) conditions. Dashed lines indicate actual intensities of blue light (5, 25 and 50 μmol photons m-2 s-1) that can induce the accumulation, avoidance and cold-avoidance in M. polymorpha [6,42].
Fig 2.
Chloroplasts avoid weak blue light under cold conditions.
(A) Diagram of the experimental conditions used to induce the cold-avoidance response with vertical or horizontal irradiation of blue light. After incubation under 25 μmol photons m-2 s-1 of blue light (BL25) with vertical irradiation at 5°C for 24 h, the WT gemmalings were irradiated by horizontal BL25. (B) Chlorophyll fluorescence images after irradiation of vertical or horizontal BL25 as shown in (A). The arrow indicates the light direction. (C) Diagram of the experimental conditions used to induce the accumulation response at 22°C and cold-avoidance response at 5°C with horizontal irradiation of vertically vibrating polarized weak BL. Ten-day-old WT sporelings were irradiated by the polarized weak BL (BL25) at 22°C for 12 h. The cells were transferred to 5°C, followed by incubation for 24 h and 60 h. (D) Chlorophyll fluorescence images after irradiation of the polarized weak BL as shown in (C). (E) P/A ratios calculated from chlorophyll fluorescence images shown in (D). Error bars indicate standard deviations.
Fig 3.
Cold-induced reduction of the transitional light intensity.
(A) Chloroplast positioning in WT gemmalings incubated at 5°C for 12 h with 5, 10, 15, and 25 μmol photons m-2 s-1 of blue light (BL5, BL10, BL15, and BL25, respectively). After pre-incubation of 1-day-old WT gemmalings under BL25 at 22°C for 1 h to induce the accumulation response, the cells were transferred to the indicated BL conditions at 5°C. (B) The P/A ratios, calculated from chlorophyll fluorescence images after incubation for 0, 2, 6, 8, 12, and 24 h. Error bars indicate standard deviations. (C) Diagram of experimental conditions used to demonstrate the cold-induced reduction of the transitional intensity. After pre-incubation under BL25 at 5°C for 24 h, WT gemmalings were transferred to BL5 or dark and incubated at 5°C for 24 h. (D) Representative chlorophyll fluorescence images of WT gemmalings after pre-incubation (BL25) and incubation under BL5 (from BL25 to BL5) or dark (from BL25 to Dark) as shown in (C).
Fig 4.
Overexpression of Mpphot-Citrine reduces the transitional light intensity.
(A) Immunoblot analysis of WT and Mpphot-Citrine overexpression lines (35S::Mpphot-Citrine/WT: OX1, OX2, and OX3) with anti-Mpphot antibody. White and black arrowheads indicate Mpphot-Citrine and Mpphot, respectively. Rubisco large subunit (RBCL) was shown as a loading control. (B) Representative images of chlorophyll fluorescence in WT and Mpphot-Citrine overexpression lines after the incubation under BL25 at 22°C for 12 h. (C) P/A ratios calculated from the chlorophyll fluorescence images shown in (B). (D) Gel-mobility shift assay to detect BL-dependent phosphorylated forms of native Mpphot and Mpphot-Citrine in WT and Mpphot-Citrine overexpression lines (OX1, OX2, and OX3) at 22°C. Four-day-old gemmae were incubated under dark condition for 72 h or BL25 condition for 12 h. Immunoblot analysis was performed with anti-Mpphot antibody. Magenta arrowheads indicate the band positions of BL-dependent phosphorylated forms (WT, OX1, OX2, and OX3) of Mpphot-Citrine (upper) and native Mpphot (lower). Black arrowheads indicate the band positions of dark-adapted (WT and OX1) and dephosphorylated (OX3*) forms of Mpphot-Citrine (upper) and native Mpphot (lower). The asterisk indicates the dephosphorylated OX3 sample by phosphatase treatment (10 units of FastAP thermosensitive alkaline phosphatase, Thermo Fisher Scientific) [6]. (E) Representative images of chlorophyll fluorescence in WT and Mpphot-Citrine overexpression lines (OX1 and OX2) after incubation under BL15, BL10 or BL5 at 22°C for 12 h. (F) P/A ratios calculated from the chlorophyll fluorescence images shown in (E). (G) Confirmation of the reduction of transitional light intensity in OX2 under cold conditions. In the 1-day-old gemmalings from OX2, the accumulation response was induced under BL5 at 22°C (left panel). The cold-avoidance response was induced under BL5 at 5°C for 24 h (right panel). (H) Time-course of the cold-avoidance response induced in OX2 under BL5 at 5°C. The P/A ratios were calculated from the chlorophyll fluorescence images after incubation for 0, 6, 12, and 24 h.
Fig 5.
Increased Mpphot expression is not required for the induction of the cold-avoidance response.
(A) Immunoblot analysis of endogenous Mpphot in 4-day-old WT gemmalings (white light for 3 days and BL25 for 24 h at 22°C), incubated under BL25 for 0 h (22°C), and 3 h, 6 h and 9 h at 5°C. Histone H3 protein is shown as a loading control. (B) Representative chlorophyll fluorescence images of WT gemmalings incubated under the same light and temperature conditions as shown in (A). (C) P/A ratios calculated from chlorophyll fluorescence images after incubation for 0 h (22°C), and 3 h, 6 h and 9 h at 5°C. Error bars indicate standard deviations. (D) Immunoblot analysis of activity of CaMV35S promoter in M. polymorpha under cold conditions. A transformant expressing Citrine under the control of CaMV35S promoter (35S::Citrine/WT) was used. Coomassie Brilliant Blue (CBB)-stained Rubisco large subunit (RBCL) was used as a loading control. (E) Chlorophyll fluorescence images showing the cold-avoidance response in complementation lines (35S::Mpphot-Citrine/MpphotKO). After incubation under BL25 at 22°C, the gemmalings were transferred to 5°C under the same light conditions.