DEV030585 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Figure S1 -

  Fig. S1. Detailed protocol of MeJA/wound treatment. Since MeJA strongly inhibits seed germination and seedling growth, plants were initially allowed to grow without any treatment. On 6 DAG, a small strip of filter paper was placed in the center of a plate. A cap of a 1.5 ml tube was used to avoid direct contact between filter paper and agar surface. MeJA (25 nmol, dissolved in methanol) was absorbed onto the filter paper to allow vaporization. A higher concentration of MeJA (250 nmol/plate) was also used to ensure a JA response in a short period. The plates (9 cm diameter, 2 cm height, containing 25 ml of solidified MS medium) were sealed with Micropore surgical tape (3M Health Care). Note that the composition of MS medium affects the plant’s response to JAs and, especially, we recommend the use of 0.5% gellan gum (Wako Pure Chemical Industries), not agar, as the gelling agent. The optimal sucrose concentration is 1.5%.

• Supplemental Figure S2 -

  Fig. S2. Wound-induced trichome formation in gl1-2 is dependent on JAs. (A) 16-DAG double mutants (aos gl1-2, coi1-1 gl1-2, jar1-1 gl1-2, myc2-1 gl1-2) grown under standard conditions (left column) or wounded on 8 DAG (right column). (B) Quantification of trichome number per leaf with or without wounding (mean±s.e. of at least 12 plants).

• Supplemental Figure S3 -

  Fig. S3. Time-course analysis of JA-induced trichome formation. Cryoscannning electron micrograph of gl1-2 (left) and urm9 gl1-2 (right) treated with MeJA on 6 DAG and sampled at 0, 24, 48 and 96 hours post-treatment. (A,B) Zero hours post MeJA treatment. First and second true leaves are glabrous (and remain glabrous thereafter). (C,D) Twenty-four hours post MeJA treatment. Third and fourth true leaves are emerging. (E,F) Forth-eight hours post MeJA treatment. Third and fourth true leaves start to initiate trichomes in gl1-2 but not in urm9 gl1-2. (G,H) Ninety-six hours post MeJA treatment. gl1-2 continues to produce dense trichomes on new leaves. urm9 gl1-2 occasionally forms trichomes with the normal three branches. (I,J) Ninety-six hours post mock treatment. Both gl1-2 and urm9 gl1-2 are glabrous. Scale bar: 500 µm.

• Supplemental Figure S4 -

  Fig. S4. GL1 expression is not upregulated by MeJA. (A) GL1::GUS expression in gl1-2 (low-magnification view of Fig. 3C). Left, control plants; right, MeJA-treated plants. Three representative plants are shown. Scale bar: 500 µm. (B) Phenotype of 35S::GL1. Ectopic overexpression of GL1 blocks normal trichome formation as well as MeJA-induced trichome formation.

• Supplemental Figure S5 -

  Fig. S5. Root and seed epidermal cell differentiation in urm9 and urm23. (Upper row) Root epidermal patterning. urm9 and urm23 show the normal, striped root hair pattern, whereas gl2-3125 and ttg1-213 form root hairs from all cell files. (Middle row) Biosynthesis of proanthocyanidin tannin (brown pigment) in the seed coat. urm9, urm23 and gl2-3125 seeds are brown, whereas ttg1-213 seeds are light yellow. (Lower row) Secretion of mucilage from water-imbibed seed coat. Seed mucilage was stained with 0.01% Ruthenium Red solution. urm9 and urm23 seeds secrete mucilage normally, whereas ttg1-213 seeds almost lack mucilage and gl2-3125 seeds completely lack mucilage. Scale bars: 1 mm.

• Supplemental Figure S6 -

  Fig. S6. Map-based cloning of URM23. (A) Mapping of urm23. urm23 was tightly linked with the SSLP markers MYJ24 and NGA139 in the upper arm of chromosome 5. The TTG1 gene is located between MYJ24 and NGA139 and is especially close to NGA139. Sequencing of the TTG1 locus from the urm23 genome revealed a G-to-A point mutation in the first exon of TTG1. (B) The protein structure of multiple TTG1 variants. Blue box indicates the conserved WD40 repeat. (C) Allelism test between urm23 and ttg1. The urm23 mutation behaves as dominant in a gl1-2 homozygous background, but behaves as recessive in a gl1-2/+ heterozygous background. Therefore, we crossed urm23 gl1-2 and ttg1-213 and found that the resultant F1 plants (urm23/ttg1-213 gl1-2/+) were glabrous, showing non-complementation of urm23 and ttg1-213. (D) MeJA response of ttg1-213. ttg1-213 gl1-2 lacks trichome induction as well as anthocyanin induction in response to MeJA treatment. ttg1-213/+ gl1-2 lacks trichome induction but shows intact anthocyanin induction in response to MeJA.

• Supplemental Figure S7 -

  Fig. S7. Map-based cloning of URM9. (A) Mapping of urm9. urm9 was delimited to a 161 kb region in the bottom arm of chromosome 2, flanked with CAPS markers F16D14-3 and F20M17-1. This region overlaps with the map position of RTN, a major QTL controlling the natural variation in trichome density. According to the TAIR7 genome annotation (http://www.arabidopsis.org), we sequenced the coding region of all 49 gene models (At2g31320 to At2g31780), except for a LINE retrotransposon (At2g31520) predicted in this region. As a result, two point mutations were found: one in At2g31480 and the other in At2g31660. The C-to-T point mutation in the single exon of At2g31480 results in a Ser186 to Leu substitution. The G-to-A point mutation in the sixth exon of At2g31660 generates a premature stop codon (Trp256 to stop). (B) Transgenic complementation of At2g31480 and At2g31660. Genomic fragments of 4.1 kb or 9.3 kb (blue lines in A) containing full-length At2g31480 and At2g31660, respectively, were transformed into urm9 gl1-2 and tested for the rescue of MeJA-induced trichome formation. (C) Allelism test between urm9 and T-DNA insertion alleles of At2g31480 or At2g31660. We crossed urm9 gl1-2 with 31480-1, 31480-2 or sad2-2 to generate urm9/31480-1 gl1-2/+, urm9/31480-2 gl1-2/+ or urm9/sad2-2 gl1-2/+, respectively. Shown is a quantification of the trichome number per leaf in the first/second or fifth true leaf (mean±s.e. of at least 12 plants).

• Supplemental Figure S8 -

  Fig. S8. Subcellular localization of GL3-2xGFP in ttg1-10 and gl1-S92F. Confocal image of leaf adaxial epidermal cells of ttg1-10 GL3::GL3-2xGFP (left) and gl1-S92F GL3::GL3-2xGFP (right), both of which show abnormal subnuclear speckles of the GL3-2xGFP fusion protein. Scale bars: 10 µm.

• Supplemental Figure S9 -

  Fig. S9. MeJA-responsive expression of GL3::GL3-2xGFP in the GL1 background. Epifluorescence images taken under the same setting to compare the intensity of GFP fusion expression. Shown are representative images from at least 20 plants examined. (A-D) GL3::GL3-2xGFP. (E-H) urm9 GL3::GL3-2xGFP. Plants were either left untreated (A,B,E,F) or treated with MeJA on 6 DAG (C,D,G,H). GFP fluorescence was observed and photographed on 7 DAG (24 hours post-treatment). (Left) GFP fluorescence (green) and chlorophyll autofluorescence (red). (Right) GFP fluorescence (green).

• Supplemental Figure S10 -

  Fig. S10. Subcellular localization of GL3-2xGFP in the GL1 background. (A,C,E) Ubiquitous expression of GL3::GL3-2xGFP in young leaves of the wild type (A) and urm9 GL1 single mutant (C,E). The green signal is GFP-specific fluorescence and the red signal is chlorophyll autofluorescence. (B,D,F) Higher magnification view of A,C,E (boxed regions), showing the subcellular localization of GL3-2xGFP in the adaxial epidermal cells. In most urm9 GL1 leaves, GL3-2xGFP was evenly distributed in the nucleus as in wild type (D) and the subnuclear speckles were only rarely observed (F).