PaFD, a PaFT1-interacting bZIP protein.

<p>A, Comparison of PaFD with other FD proteins from <i>Arabidopsis</i> and rice. Green boxes indicate bZIP domain and the number in the parenthesis is the length of each polypeptide. B, PaFT1 interacts with PaFD in the yeast system. PaFD also interacts with FT proteins from other species such as <i>Arabidopsis</i> (AtFT, AtTSF), rice (Hd3a, RFT1) and <i>Oncidium</i> orchid (OnFT). PaFT1Y86H indicates mutant form of PaFT1 and PaFDΔN1-53, PaFD T225A, S226A, S227A indicates N-terminal deletion and mutant forms of PaFD, respectively. The interaction between AtFT and AtFD is a positive control. C, YFP:PaFT1 fusion proteins in <i>Arabidopsis</i> cell. D, CFP:PaFD fusion proteins in <i>Arabidopsis</i> cell. E, Merged image of YFP:PaFT1 and CFP:PaFD proteins in <i>Arabidopsis</i> cell. F, G, H and I, BiFC assays in <i>Arabidopsis</i> cells. Plasmids for YFPn:PaFT1 and YFPc:PaFD expression were introduced into <i>Arabidopsis</i> cells, simultaneously. J, L and M, BiFC assays in <i>Phalaenopsis</i> cells. K, NLS:RFP was used for nuclear localization marker. Bar is 40 ìm in E, G, I and 20 ìm in M.</p

Phloem-specific expression of <i>PaFT1</i> in <i>Arabidopsis</i> and overexpression of <i>PaFT1</i> in rice drive early flowering.

<p>A, Comparison of flowering time between transgenic plants containing p<i>SUC2</i>:<i>PaFT1</i> and wild type plants. Thirty independent T1 plants were used for each genotype. Arrows represent the mean value of total leaf number in each genotype. P ≤ 0.0001 (Student’s <i>t</i>-test). B and C, <i>PaFT1</i> driven by phloem-specific or shoot apex-specific promoters rescues the late flowering phenotype of <i>Arabidopsis ft</i> null mutant, <i>ft-10</i>. This activity is at least dependent on Tyr-86 residue, one of the conserved amino acids among FT proteins from various plant species. D and E, Ectopic expression of <i>PaFT1</i> in rice also caused early flowering (Dongjin cultivar, grown under SD condition). Magnified panicles are shown in the box of E and flowering time data is shown in D. TLN means total leaf number.</p

The VIGS of <i>PaFT1</i> exhibits delayed spiking.

<p>A and B, Compared with control lines for the VIGS of <i>GUS</i> gene, the VIGS of <i>PaFT1</i> lines show late flowering. B, Spiking time was measured in thirty independent lines for VIGS of <i>PaFT1</i> together with twenty lines for VIGS of <i>GUS</i> and five non-treated lines. We counted the first day of transferring the orchids to the low temperature condition (23°C / 20°C) after VIGS treatment as day one for spiking. C, Reduced expression of endogenous <i>PaFT1</i> in the VIGS lines of <i>PaFT1</i>. NT and GC mean non-treated and <i>GUS</i> control (VIGS of <i>GUS</i> gene), respectively.</p

Growth and flowering of <i>P</i>.<i>aphrodite</i> subsp. <i>formosana</i> with the expression of <i>PaFT1</i>.

<p>A, Floral buds at different developmental stages and the structure of flower. S: sepal, P: petal, L: lip, C: column, Pe: pedicel. Bar is 1 cm. B, Spiking and flowering of <i>P</i>. <i>aphrodite</i> subsp. <i>formosana</i> under LD and SD conditions at constant 23°C. C, Under LD conditions, low temperature treatment is essential to induce inflorescence of <i>P</i>.<i>aphrodite</i> subsp. <i>formosana</i>. Day and night temperatures are shown in parenthesis. D, Spatial expression of <i>PaFT1</i> in <i>P</i>. <i>aphrodite</i> subsp. <i>formosana</i>. Materials for RNA extraction were harvested from six to eight plants. Bud 1, bud 2 and bud 3 indicate the B1, B2 and B3, respectively in A. Spike 1, spike 2 and spike 3 indicate ≤ 3 cm, 3–10 cm and ≥ 10 cm in length, respectively. The 3rd, 4th and 5th leaves were used for the leaf RNA extraction. E, Daily oscillation of <i>PaFT1</i> expression under LD and SD conditions. In each time point, leaves of 4 plants (18 months old) were harvested for RNA extraction. F, The effect of ambient temperature on <i>PaFT1</i> expression. LT; low temperature (23°C/20°C), HT; high temperature (28°C/25°C). Thirty six mature plants (34-month old as the stage 4) were grown at HT and then sixteen plants were transferred to the LT conditions. All leaves of four plants were used for the analysis of <i>PaFT1</i> expression, at each time point. All the samples were harvested at the end of light (ZT 16). Two independent experimental results showed similar expression patterns.</p

Spatiotemporal expression pattern of <i>PaFD</i> and its functional activity in <i>Arabidopsis</i>.

<p>A, Developmental stages of floral buds and spikes are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134987#pone.0134987.g001" target="_blank">Fig 1</a>. B, <i>PaFD</i> expression in a developing spike by <i>in situ</i> hybridization. The magnified axillary floral meristem in the emerging spike (≤ 3 cm in length) is in the box (left panel) and the middle panel is a negative control with a sense probe. <i>PaFD</i> transcript was detected in the floral meristems in the developing spike (3–10 cm in length, right panel). Bar is 100 ìm. C, Expression pattern of <i>PaFD</i> in different developmental stages. The stage 1 (S1) is 16-month and the S2 is 20-month old stages of the orchid in the flasks. S3 and S4 show 26-month and 34-month old stages of the orchid in the pots, respectively. Bar is 1 cm. D, Flowering phenotypes of wild type, <i>fd</i> mutant (<i>fd-3</i>), and two independent homozygous transgenic plants expressing <i>PaFD</i> under the control of <i>Arabidopsis FD</i> promoter in <i>fd-3</i> background. E, Flowering time of the each genotype shown in D. Twelve individuals for each genotype were used for flowering time measurement and <i>PaFD</i> expression is detectable only in the transgenic plants.</p

Phloem-specific expression of <i>PaFT1</i> reduces the late flowering effect by overexpression of <i>SVP</i> encoding an ambient temperature-dependent floral repressor.

<p>A and D, p<i>SUC2</i>:<i>PaFT1</i> retards the effect of late flowering by p<i>35S</i>:<i>SVP</i>. B and C, The expression of <i>SOC1</i> and <i>FUL</i> in each genotype shown in A. Relative expression was presented compared with that of Col WT. E, A model showing the effect of <i>PaFT1</i> expression in <i>Arabidopsis</i> flowering.</p

Ectopic expression of <i>PaFD</i> causes early heading in rice.

<p>A and B, The vector control plant is a transgenic rice plant containing an empty vector. Expression level of two rice <i>AP1</i> homologues, <i>OsMADS14</i> and <i>OsMADS15</i> is high compared with the control. VC indicates vector control. C, Flowering time (heading date) of the transgenic rice plants overexpressing <i>PaFD</i> compared with the control containing empty vector. Eight to twelve individual plants per each line were used for heading date measurement.</p

Genomic <i>PaFT1</i> partially complements <i>Arabidopsis ft</i> mutants.

<p>A, Genomic structure of <i>PaFT1</i>. <i>AtFT</i> is an <i>Arabidopsis FT</i> and <i>Hd3a</i> is a rice <i>FT</i> homologue. Filled boxes indicate coding sequences and the lines between boxes represent introns. Numbers represent the length of nucleotides in coding regions and introns (bp). B and C, 6-kb genomic clone of <i>PaFT1</i> containing its 4-kb promoter region partially rescues the late flowering phenotype of <i>Arabidopsis ft</i> null mutants. C, Flowering time was measured with twenty five individual T1 plants containing the genomic clone in the <i>ft</i> mutant background and twenty <i>ft-10</i> plants. Arrows represent the mean value of total leaf number in each genotype. P ≤ 0.0005 (Student’s <i>t</i>-test).</p

Time-dependent photoconversion of PIN2-Dendra2 fusion protein.

<p>(<b>A</b>) The same root was analyzed for green and red fluorescence before (time 0) and after 3 s and 20 s conversion. (<b>B</b>) Efficiency of photoconversion depends on the time of illumination with blue-violet light. Green and red fluorescence signals were collected and the intensities of 150 transversal membranes within 5 roots were assessed with the ImageJ software. The interrupted line with open circles represents changes in red signal intensities, the solid line with closed circles represents decreasing in green signal intensities, bars represent SE. (<b>C</b>) Emission spectra of green and red signals of unconverted and photoconverted membranes were analyzed in three roots (represented by three lines in the graph). The HFT UV/488/543/633 was used as a beam splitter. Each point on the graph with SE bar represents the mean of 30 transversal membranes.</p

Jasmonates influence PIN2 plasma membrane dynamic.

<p>Both JA and MeJA at the concentrations 5 µM and 50 µM affect green signal intensity in transversal plasma membranes of unconverted samples (p≤0.01 for 5 µM JA and 5 µM MeJA, p≤0.001 for 50 µM JA and 50 µM MeJA,). Differences were significant at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA, at time point 6 h (p≤0.05) and 12 h (p≤0.01) for 5 µM MeJA, and at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 50 µM MeJA. For others differences were statistically not significant. Both jasmonates at the concentrations examined slowed-down the disappearance of red signals from the membrane (labeled as converted red; for each jasmonate and concentration p≤0.001) For both jasmonates used at a concentration of 5 µM differences were significant at time point 1.5 h (p≤0.01), 3 h (p≤0.001) and 6 h (p≤0.001), for 50 µM JA at time point 3 h (p≤0.05) and 6 h (p≤0.001), for 50 µM MeJA at time point 3 h (p≤0.05) and 6 h (p≤0.05). For others, the differences were statistically not significant. Both jasmonates at the concentrations examined delayed membrane green signal recovery (labeled as converted green; for each jasmonate and concentration p≤0.001)). Differences were significant at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.01) and 12 h (p≤0.01) for 5 µM MeJA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA and 50 µM MeJA. For others, the differences were statistically not significant.</p