Phototropins of the moss Physcomitrella patens function as blue-light receptors for phototropism in Arabidopsis

Abstract

Four phototropin genes (PHOTA1, PHOTA2, PHOTB1, PHOTB2) have been isolated in the moss Physcomitrella patens. These genes encode phototropins that mediate blue-light-induced chloroplast movement. However, the individual functions of these phototropins, including the function of mediating blue-light-induced phototropism, remain unclear. To elucidate the individual functions of P. patens phototropins, each of these phototropin genes was expressed in a phototropin-deficient mutant of Arabidopsis (phot1-5 phot2-1). In addition, fluorescence of GFP fused to these phototropins was examined to determine the subcellular localization of each phototropin. Our results demonstrate that all four P. patens phototropins mediate blue-light-induced phototropism and are associated with the plasma membrane in Arabidopsis. Abbreviations GFP: green fluorescent protein; Pp_phot: Physcomitrella patens phototropin.

Keywords: Phototropism; Physcomitrella; blue light; moss; phototropin; plasma membrane; red light.

Figure 1.

Expression of Pp_phot:GFP in transgenic Arabidopsis (a) Schematic illustration of the binary vector construct used to make transgenic Arabidopsis expressing Pp_phot:GFP fusion protein. CaMV 35S Pro: cauliflower mosaic virus 35S promoter; Nos Ter: nopaline synthase terminator; Km^r: kanamycin resistant gene cassette(b) Immunoblot analysis of Pp_phot:GFP protein. Crude protein extract (30 μg) prepared from dark-grown transgenic Arabidopsis seedlings expressing Pp_phot:GFP (photA1:GFP-21, photA2:GFP-16, photB1:GFP-13, or photB2:GFP-6) was probed with the anti-GFP antibody. An asterisk on the left indicates a nonspecific band that had cross-reacted with GFP antibody. After chemiluminescence detection, the polyvinylidene difluoride (PVDF) membrane was stained with Coomassie brilliant blue (CBB) to visualize the amount of protein loaded in each lane (CBB stain).

Figure 2.

Phototropic responses of transgenic Arabidopsis seedlings Wild-type, phot1-5 phot2-1, and transgenic Arabidopsis lines expressing Pp_phot:GFP were grown in the dark for 3 days, and irradiated by unilateral blue light (5 μmol m⁻² s⁻¹) for 16 hr before imaging. All of the transgenic lines were heterozygous for the transferred gene. Scale bars, 1 cm. (left). The percentage of seedlings with hypocotyl angles relative to vertical is shown in 6 classes: less than −30, −30 ~ −10, −10 ~ 10, 10 ~ 30, 30 ~ 50, and more than 50°. A positive value indicates the degree of curvature which shows bending toward the light source. Hypocotyl curvatures were measured in at least 74 seedlings (right).

Figure 3.

Subcellular localization of the Pp_phot:GFP in transgenic Arabidopsis seedlings A single optical section of Pp_phot:GFP expressing transgenic Arabidopsis, which was stained with the FM 4–64, was captured by confocal imaging. Upper hypocotyl epidermal cells of the 3-day-old dark-grown seedlings were observed. Scale bars, 20 µm.

Figure 4.

Multiple alignment of the amino-acid sequences of the C-terminal region in phototropins Residues conserved in all sequences are marked with an asterisk and highly or moderately similar residues are marked with dots or a dot, respectively. The short amino-acid stretches that were suggested to be important for phototropin’s membrane association in Arabidopsis are enclosed in red boxes. Within the stretches region, conserved or similar amino acid residues are highlighted with blue or green background, respectively. Pp (Physcomitrella patens); Mp (Marchantia polymorpha); At (Arabidopsis thaliana).