Red Light-Induced Phytochrome Relocation into the Nucleus in Adiantum capillus- veneris  Hidenori Tsuboi, Sachihiko Nakamura, Eberhard Schäfer, Masamitsu Wada  Molecular Plant  Volume 5, Issue 3, Pages 611-618 (May 2012) DOI: 10.1093/mp/ssr119 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Fluence–Response Relationship for Spore Germination. (A) Spores imbibed for 5 d under darkness were irradiated with a R microbeam (255 × 440 μm) with 4.2 W m−2 for various periods of time (from 3 to 240 s). Arrow indicates the fluence (252 J m−2) used for the following experiments. (B) Fluence–response relationship for FR reversibility on the R-induced spore germination. Whole spore was irradiated with 4.2 W m−2 of R microbeam (255 × 440 μm) followed by an FR microbeam (255 × 440 μm) irradiation for various periods of time (from 3 to 480 s). Arrow indicates the fluence (1440 J m−2) used for the following experiments. (C) Escape response from FR reversibility in R-induced spore germination. Spores were irradiated with an R microbeam (255 × 440μm; 4.2 W m−2 for 60 s) and then incubated in the dark for various periods of time from 0 to 12 h. Then, the spores were irradiated with an FR microbeam (255 × 440 μm; 6.0 W m−2 for 240 s) and incubated in the dark for 5 d. Each point represents the mean ± SE determined from two or three experiments. More than 100 spores were counted in each experiment. Molecular Plant 2012 5, 611-618DOI: (10.1093/mp/ssr119) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Translocation of Phytochrome Response from Cytoplasm to a Nuclear Region. (A) Whole or either a nucleus or a part of cytoplasmic area of a spore (inset) imbibed for 5 d under darkness was irradiated with R microbeam (mR: circle, 12 μm in diamater; 4.2 W m−2 for 60 s). Germination rate was counted 5 d after the light treatments. Germination rate without light treatment was also shown. (B) Either nucleus or a part of cytoplasmic area was irradiated with FR microbeam (mFR in the upper panel: circle, 12 μm in diameter; 6.0 W m−2 for 240 s) just after (white bars) or 2 h (gray bars) after R irradiation (wR: 4.2 W m−2, 60 s). Others are the same as in (A). (C) Whole spore was irradiated with R (wR: 4.2 W m−2, 60 s), incubated for 2 h in darkness, and then irradiated whole spore with FR (wFR: 6.0 W m−2, 240 s). Finally, either nucleus or a part of cytoplasmic area was irradiated with an R microbeam (mR: circle, 12 μm in diameter; 4.2 W m−2 for 60 s). Each point represents the mean ± SE determined from two or three experiments. More than 20 spores were counted in each experiment. Other details are the same as in Figure 1. Molecular Plant 2012 5, 611-618DOI: (10.1093/mp/ssr119) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phytochrome Transcription Levels under Different Light Conditions. The dry spores (Dry), the spores imbibed under darkness for 5 d (5D), the spores imbibed under darkness for 5 d and then cultivated under R for 3 d (3R), and protonemata incubated under R for 8 d after sowing (8R) were tested. RT–PCR analyses were performed using primers specific for AcPHY1, AcPHY2, and AcCRY2. Molecular Plant 2012 5, 611-618DOI: (10.1093/mp/ssr119) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Intracellular Distribution of phy–GUS Fusion Proteins in Germinating Cells. (A) Representative distribution patterns of phy1–GUS and phy2–GUS fusion proteins under R or darkness were taken by Nomarski optics. (B) Co-localization of GUS activity (upper panels) and DAPI staining (lower panels) were shown in phy2–GUS fusion proteins (left panels) and GUS–NLS fusion proteins (right panels). Note that the phy2–GUS under R and GUS–NLS proteins under darkness localized in the nucleus (white arrow heads). (C) Three different categories of GUS staining taken by Nomarski optics (upper panels) and staining levels expressed by color scale (lower panels): stained only one restricted area (N), stained whole area with one well-stained area (C+N), stained evenly around whole area (C). (D, E) Histogram of intracellular distribution of phy1–GUS (D) and phy2–GUS (E) proteins under the dark (black bars) and R (white bars) conditions. Bars: 10 μm. Molecular Plant 2012 5, 611-618DOI: (10.1093/mp/ssr119) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions