Data_Sheet_1.DOCX

<p>Plant Rho-like GTPases (ROPs) are switch-like proteins which play essential roles in controlling cell polarity development and cellular activities. ROPs are regulated by many factors, such as auxin, light, and RopGEFs and RopGAPs proteins. However, it has not been reported yet whether small molecules play a role in the regulation of ROP activity. Here, we showed that AtROP6 specially bound to a phospholipid, phosphatidylglycerol (PG), by the protein-lipid overlay and liposome sedimentation assays, and further MST assay gave a dissociation constant (Kd) of 4.8 ± 0.4 μM for binding of PG to His-AtROP6. PG profile analysis in Arabidopsis revealed that PG existed both in leaves and roots but with distinctive fatty acyl chain patterns. By evaluating AtROP6 activity using RIC1 effector binding-based assay, we found that PG stimulated AtROP6 activity. In the FM4-64 uptake experiment, PG inhibited AtROP6-mediated endocytosis process. By evaluating internalization of PIN2, PG was shown to regulate endocytosis process coordinately with NAA. Further root gravitropism experiment revealed that PG enhanced the AtROP6-mediated root gravity response. These results suggest that the phospholipid PG physically binds AtROP6, stimulates its activity and influences AtROP6-mediated root gravity response in Arabidopsis.</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

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

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 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

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

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> 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

<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