Figure 1.
Schematic representation of the XVE system.
Schematic representations of pER8 (A: modified from [22]), pPGX (B), pLGZ2 (C) vectors. RB: right border [22], PG10-90: a G10-90 synthetic promoter [32], TrbcSE9: a Pea rbcS E9 terminator [33], Pnos: Agrobacterium nopaline synthase promoter [17], HPT: the hygromycin phosphotransferase gene [17], Tnos, an agrobacterium nopaline synthase terminator [17], LexA:Pm35S: eight copies of LexA operators [22] connected to CaMV minimal 35S promoter [23], MCS: multi cloning site including XhoI, AscI, ApaI, PacI, and SpeI. TrbcS: a TrbcS terminator [34], LB: left border [22], PIG1R and PIG1L: DNA fragments for homologous recombination to the PIG1 putative neutral site [14], GW: the Gateway cassette rfA containing the chloramphenicol resistance gene and the ccdB gene flanked by attR1 and attR2 sites (Life Technologies), Tpea3A: a Tpea3A terminator [34], PGX: one of GX promoters, XVE: the chimeric gene with a LexA-binding [28,29], a VP16 activator [30], and an estrogen receptor domain [31], aphIV: the hygromicin phosphotransferase expression cassette [35,36], PTA2R and PTA2L: DNA fragments targeting to the PTA2 putative neutral site, loxP: the sequences for site-specific recombination by Cre recombinase [55], zeo: the bleomycin resistance protein expression cassette [36]. Backbone of pER8 is pZP200 [22]. Backbones of pPGX and pLGZ2 are pBluescriptII [49].
Figure 2.
Spatial expression patterns of NGG induced by P. patens XVE system.
Fluorescence images of protonemata (A) and gametophores (B) of GX6-NGG#63 and GX8-NGG#4 lines. Fluorescent signals were observed after 24 h inoculation in water with (+) or without (-) 1 µM β-estradiol. Yellowish color of leaf veins in GX8-NGG#4 is caused by reflection but not by NGG signals. (C) Magnified views of apexes of gametophores in GX6-NGG#63 and GX8-NGG#4 lines. Pictures in A, B, and C panels are at the same magnification. Bars = 100 µm.
Figure 3.
Amounts of NGG and TUA1 transcripts in Pm35S:NGG, Pact1:NGG, and HSP:NGG lines.
(A, B) Transcript amounts of NGG (A) and TUA1 (B) in protonemata incubated for 8 days after propagation. Three independently transformed lines harboring a single copy of each DNA fragment at the PIG1 targeting site were analyzed (Figures S4 to S6). Copy numbers of the NGG and TUA1 transcripts per pg total RNA were estimated by RT-qPCR. In HSP:NGG lines, protonemata were cultured for 8 days at 25°C and collected after 1 h incubation at 25°C (crosses) or 38°C (squares).
Figure 4.
Amounts of NGG and TUA1 transcripts in GX6-NGG and GX8-NGG lines.
(A, B) Transcript amounts of NGG (A) and TUA1 (B) in protonemata immersed in water with (+: circles) or without (-: crosses) 1 µM β-estradiol for 24 h before sampling. Three independently transformed lines harboring a single copy of each GX-NGG DNA fragment at the PIG1 targeting site were examined. Copy numbers of each NGG and TUA1 gene per pg total RNA were estimated by RT-qPCR.
Figure 5.
Dose responsiveness to β-estradiol in the GX6-NGG and GX8-NGG lines.
(A, B) Relative transcript levels of NGG in protonemata of GX6-NGG#63 (squares) and GX6-NGG#65 (triangles) line (A) and GX8-NGG#4 (squares) and GX8-NGG#16 (triangles) line (B). Protonemata were immersed in water containing different concentrations of β-estradiol for 24 h before sampling. Relative transcript levels of NGG are normalized to TUA1 levels and standardized to a normalized transcript level of protonemata without β-estradiol. Error bars indicate SD of the mean (n = 3).
Figure 6.
Time course responsiveness to β-estradiol in the GX6-NGG and GX8-NGG lines.
(A, B) Relative transcript levels of NGG in protonemata of GX6-NGG#63 (A) and GX8-NGG#4 (B) lines. Relative transcript levels of NGG are normalized to TUA1 and then standardized to the normalized transcript level of protonemata at 0 h. Protonemata were immersed in water with (+: circles) 1 µM β-estradiol and collected after 0, 1, 3, 6, 12, 24, and 48 h. As a negative control, protonema cultivated without (-: crosses) β-estradiol were collected after 0, 6, 12, and 48 h. Error bars indicate SD of the mean (n = 3).
Figure 7.
Spatial expression patterns of dually induced NGG and NmRFP1.
Fluorescence images of protonemata and a gametophore leaf of the GX6-NGG#63/LGZ2-NmRFP1#11 line. They were immersed in water containing 1 µM β-estradiol for 24 h before microscopy. Fluorescence images of NGG and chlorophyll autofluorescence (top row), NGG (second row), NmRFP1 (third row), and a merged image of NGG and NmRFP1 (bottom row) are indicated. Each picture is at the same magnification. Bar = 200 µm.
Figure 8.
Transcript amounts for dually induced NGG and NmRFP1 in transgenic P. patens lines.
(A, B, C) Transcript amounts of NGG (A), NmRFP1 (B), and TUA1 (C) in protonemata of GX6-NGG#63/LGZ2-NmRFP lines. Protonemata were immersed in water with (+: circles) or without (-: crosses) 1 µM β-estradiol for 24 h before sampling. Copy numbers of NGG, NmRFP1, and TUA1 genes per pg total RNA were estimated by RT-qPCR.