A Rice Phytochrome A in Arabidopsis: The Role of the N-terminus under red and far-red light  Kneissl Julia , Shinomura Tomoko , Furuya Masaki , Bolle Cordelia   Molecular Plant  Volume 1, Issue 1, Pages 84-102 (January 2008) DOI: 10.1093/mp/ssm010 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 The N-terminus of phytochromes. (A) Alignment of the N-terminal amino acids of different phytochromes. D, dicots; M, monocots. Serine residues are depicted in red. Ser-7 and Ser-17 of the oat phyA, which have been shown to been phosphorylated, are circled. (B) Schematic diagram of the phytochrome domains of higher plants. Serine residues that were changed to alanines in the rice PHYA construct are indicated. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Immunoblot analysis of phytochrome A levels in Arabidopsis lines expressing either the wild type rice PHYA (WT) or the PHYA SA mutation (SA). Constructs were expressed in phyB mutants or phyAphyB (phyAB) double mutants. Total protein extract was isolated from 3-week-old light-grown plants. phyA was detected with specific antibody (upper panel). The loading control is shown on the lower panel. Values are the relative amount of phyA protein and were normalized with the loading control. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Germination assays with different imbibition times (3 h, 1 d, 2 d). Germination efficiency was evaluated after pulses of R (A, C, E) or FR (B, D, F) light of different photon fluence. Note the different scales on the y-axis. Germination was tested for Ler (A, B), phyAphyB mutants (phyABC, D) and phyAphyB PHYA lines (phyAB WT; E, F). Mean values of germination were calculated. Error bars represent standard deviation. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Germination assays after 47 h of imbibition under different fluences of monochromatic light. (A) R light (660 nm). (B) FR light (726 nm). (C) B light (400 nm). (D) Photon fluence needed to reach 40% germination rates. (E) Germination rate under R (0.3 μmol m−2) and FR (75 μmol m−2) light. Germination after a saturating R-light pulse was set at 100% and relative germination rates are shown. Mean values were calculated. Error bars represent standard deviation. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Arabidopsis plants expressing rice phyA show altered inhibition of hypocotyl elongation and cotyledon unfolding under the different light conditions. (A) Overview of the phenotypes of wild-type and transgenic seedlings. One-day-old dark-grown seedlings were exposed for 3 d to continuous FR light (FRc, 0.5 μmol m−2s−1), pulses of FR light (FRp, 0.5 μmol m−2s−15 min/h), continuous R light (R, 0.3 μmol m−2s−1) or darkness (D). (B) Quantification of the hypocotyl elongation. At least three different experiments were performed. Hypocotyl elongation under darkness was set 100% and percent elongation calculated. Error bars represent standard deviation. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Arabidopsis plants expressing rice phyA show an altered VLFR of the far-red light killing response. One-day-old dark-grown seedlings were transferred for 3 d to pulsed FR light (0.5 μmol m−2s−1). Thereafter, plants were grown for 3 d in white light (60 μmol m−2s−1) before measuring the chlorophyll content per mg fresh weight. Error bars represent standard deviation. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 phyAphyB double mutants expressing rice PHYA show defects in their loss of agravitropic response under FR light. (A, B, C) Distribution of the angle of growth of phyAphyB (phyAB) mutants and Ler as controls. Two independent rice PHYA expressing lines in the phyAphyB background were evaluated. Seedlings grown on vertical plates in FR (0.5 μmol m−2s−1) light for 3 d. The growth angle of at least 40 plants produced in three independent experiments was measured. (D) Phenotype of the seedlings grown on vertical plates as described above. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 8 Adult phenotypes of phyB and phyAphyB lines expressing rice phyA. Plants were grown in long-day conditions (16 h white light, 80 μmol m−2s−1). (A) Phenotype of the different lines. (B) Measurement of the length of the petiole length. (C) Measurement of the rosette leaf sheath. (D) Flowering time determined by number of rosette leaves at bolting. At least 20 plants of each line were measured. Error bars represent standard deviation. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 9 Clustering of responses of the phyAphyB mutant expressing either WT or SA PHYA. The respective phytochrome thought responsible for the phenotype and the kind of response are indicated. FRc, continuous FR light; FRp, hourly pulsed FR light; n.d., not done. Molecular Plant 2008 1, 84-102DOI: (10.1093/mp/ssm010) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions