Summary of the <i>ft1</i> mutants in tetraploid wheat cultivar Kronos.

a<p>Mutations in the DNA (Nt, position is relative to the start codon ATG);</p>b<p>Mutations in the predicted protein (Pr) (counted from the initial methionine);</p>c<p>BLOSUM 62 scores <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094171#pone.0094171-Henikoff1" target="_blank">[36]</a>;</p>d<p>Amino acid present in other species: <i>Arabidopsis</i> (<i>At</i>), <i>Hordeum vulgare</i> (<i>Hv</i>), <i>Oryza sativa</i> (<i>Os</i>), <i>Triticum aestivum</i> (<i>Ta</i>), <i>T. monococcum</i> (<i>Tm</i>), <i>T. turgidum</i> (<i>Tt</i>), and <i>Zea mays</i> (<i>Zm</i>). All homoeologous alleles were included in the comparison for the polyploidy wheat species.</p

Transcript levels of target genes in T₀<i>Brachypodium FT1</i>_RNAi lines.

<p>(A) Comparison between the average of four transgenic plants (RNAi) and the wild type control using contrasts. (B–G) Comparison between individual transgenic lines and wild type using Dunnett’s test. (B) <i>FT1</i>, (C) <i>FT2</i>, (D) <i>FT3</i>, (E) <i>FT4</i>, (F) <i>FT5</i>, (G) <i>VRN1</i> (gene regulated by <i>FT1</i>). <i>ACTIN</i> was used as the internal control. Samples were harvested at 12∶00 p.m., approximately one week after wild type control plants began to flower. Asterisks indicate <i>P</i> values: * = <i>P</i><0.05; ** = <i>P</i><0.01; *** = <i>P</i><0.001.</p

Expression of <i>FT</i>-like genes in <i>FT1</i>_HOPE transgenic wheat.

<p>The experiment was performed on transgenic lines (<i>FT1</i>_HOPE) and the non-transgenic control Jagger. Leaf tissues were collected from two independent experiments, including plants grown under a (A) LD photoperiod for five weeks and (B) plants grown under a SD photoperiod for six weeks. <i>ACTIN</i> was used as the internal control. Samples were harvested at 10∶00 a.m. Asterisks indicate <i>P</i> values: * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001.</p

<i>FT1</i> overexpression promotes floral organogenesis.

<p>(A–C) <i>Brachypodium 35S::BdFT1:GFP</i>, (A) Shoot regeneration of non-transgenic control calli (B) direct spike formation from transformed calli and (C) rudimentary leaves associated with spikelet formation from transformed calli. (D–F) Wheat <i>Ubi::cFT-B1</i>. (D) Cluster of florets surrounded by rudimentary leaves in a transformed callus. (E) Different floral organs: lemma (Le), palea (Pa), pistil (Pi) and stamen (St). The additional organs seem to be glumes but it was difficult to determine because of the close clustering of multiple florets. (F) Anther with regions of non-viable pollen (blue color after pollen staining).</p

Transcript levels of target genes in transgenic T₁ wheat RNAi plants and the non-transgenic wheat control.

<p>(A) Comparison between the average of five transgenic plants (RNAi) and the wild type control using contrasts. (B–G) Comparison between individual transgenic lines and wild type using Dunnett’s test. (B) <i>FT1</i>, (C) <i>FT2</i>, (D) <i>FT3</i>, (E) <i>FT4</i>, (F) <i>FT5</i>, (G) <i>VRN1</i> (gene regulated by <i>FT1</i>). <i>ACTIN</i> was used as the internal control. Samples were harvested at 4∶00 p.m., when the wild type controls began to flower. Asterisks indicate <i>P</i> values: * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001.</p

<i>ft1</i> TILLING mutants.

<p>(A) Interaction plot showing the effect of <i>ft-A1</i> and <i>ft-B1</i>₂₆₃ mutations on heading time in a BC₁F₂ population segregating for both genes. Differences in heading time between the <i>FT-A1</i> alleles were significant only for the homozygous <i>ft-B1</i>₂₆₃ mutant plants. (B) Transcript levels of <i>FT-A1</i> and <i>FT-B1</i> homoeologs in three-week-old wild type Kronos. The expression level of <i>FT-B1</i> was significantly higher than that of the <i>FT-A1</i> (<i>P</i> = 0.017). <i>ACTIN</i> was used as an internal control. Samples were harvested at 10∶00 a.m. Asterisks indicate <i>P</i> values of Student’s <i>t</i>-tests: * = <i>P</i><0.05.</p

Phylogenetic analysis of <i>FT</i>-like genes in temperate grasses.

<p>Phylogenetic analysis was performed using the full-length proteins. A neighbor-joining tree was constructed using pairwise deletions and 1,000 bootstrap iterations with the program MEGA 5.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094171#pone.0094171-Tamura1" target="_blank">[52]</a>. The scale bar 0.05 represents 5% base substitution. Bootstrap numbers larger than 50 are shown in the respective nodes. To simplify the tree, only the wheat A-genome homoeologs of wheat were included. Accession information: <i>BdFT1</i> (<i>Bradi1g48830</i>), <i>BdFT2</i> (<i>Bradi2g07070</i>), <i>BdFT3</i> (<i>Bradi2g49795</i>), <i>BdFT4</i> (<i>Bradi1g38150</i>), <i>BdFT5</i> (<i>Bradi2g19670</i>), <i>BdFT6</i> (<i>Bradi3g08890</i>), <i>HvFT1</i> (DQ100327), <i>HvFT2</i> (DQ297407), <i>HvFT3</i> (DQ411319), <i>HvFT4</i> (DQ411320), <i>HvFT5</i> (EF012202), <i>HvFT6</i> (morex_contig_54196), <i>OsFTL1</i> (<i>Os01g11940</i>), <i>OsFTL2/Hd3a</i> (<i>Os06g06320</i>), <i>OsFTL3/RFT1</i> (<i>Os06g06300</i>), <i>OsFTL8</i> (<i>Os01g10590</i>), <i>OsFTL10</i> (<i>Os05g44180</i>), <i>OsFTL12</i> (<i>Os06g35940</i>), <i>OsFTL13</i> (<i>Os02g13830</i>), <i>TaFT-A1</i> (CD881060), <i>TaFT-A2</i> (BT009051), <i>TaFT-A3</i> (IWGSC_1AL_913428), <i>TaFT-A4</i> (IWGSC_2AS_5252557), <i>TaFT-A5</i> (IWGSC_5AL_2803506), <i>TaFT-A6</i> (IWGSC_6AS_4388307). <i>HvFT6</i> sequence is from the International Barley Sequencing Consortium (IBSC, <a href="http://webblast.ipk-gatersleben.de/barley/" target="_blank">http://webblast.ipk-gatersleben.de/barley/</a>).</p

Heading date and floral characteristics of wheat <i>FT1</i>_RNAi transgenic plants.

a<p>The ten T₀ lines include ‘9′, ‘78A’, ‘116’, ‘140D’, ‘152E’, ‘157B’, ‘1381’, ‘1382’, ‘1384’ and ‘1828’;</p>b<p>The three T₁ lines include ‘157B’, ‘1381’ and ‘1382’;</p>c<p>The first and second florets are normal, others deficient;</p>d<p>Stigmas are bifid and feathery but open less widely than in the wild type.</p

Silencing of <i>FT1</i> by RNAi delays heading time.

<p>(A) Heading was prevented in transgenic <i>FT1</i>_RNAi<i>Brachypodium</i> and (B) delayed in <i>FT1</i>_RNAi transgenic wheat. (C) Wheat transgenic plants at booting stage. (D) Some spikes had difficulty in emerging from the leaf sheath (leaf sheath opened manually in this picture). (E) Complete floral organs from transgenic wheat flowers (stigmas failed to open in some transgenic plants).</p