Table 1.
Primer sequences used in this study.
Figure 1.
Amino acid sequence and phylogenic tree of CPC-like R3 MYB proteins.
(A) Sequence alignment of SlTRY (Solyc01g095640.1.1), TRY (AC007288) and CPC (FJ268773). Shaded letters indicate identical residues. R3 MYB domains are indicated by a line above the sequences. (B) Phylogenic tree based on deduced amino acid sequences of CPC-like R3 MYB proteins [SlTRY, TRY, CPC, ETC1 (NM100020), ETC2 (FJ972652), ETC3/CPL3 (AB264292), TCL1 (FJ972675), TCL2/CPL4 (FJ972681), Os1g43180 and Os1g43230] were aligned with a multiple alignment program (Genetyx ver. 16.0.2 software, Genetyx, Tokyo, Japan), and a dendrogram was created using clustering with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Branch length indicates relative evolutionary distances. Numbers above branches are genetic distances based on 10,000 bootstrap replicates. Distances are shown as the p-distance.
Figure 2.
Amino acid sequence and phylogenic tree of bHLH proteins.
(A) Sequence alignment of SlGL3 (Solyc08g081140.2.1), GL3 (AF246291) and EGL3 (NM20235). Shaded letters indicate identical residues. bHLH regions are indicated as line above the sequences. (B) Phylogenic tree based on deduced amino acid sequences of bHLH proteins [SlGL3, GL3, EGL3, TT8 (AJ277509), AtMYC1 (AF251697), AtbHLH003 (AF251688), AtbHLH004 (AF251689), AtbHLH005 (AF251690), AtbHLH006 (X99548), AtbHLH013 (AY120752), AtbHLH014 (AJ619812), AtbHLH017 (AY094399), AtbHLH018 (AF488562), AtbHLH025 (AF488567) and AtbHLH028 (AF252636)] aligned with a multiple alignment program (Genetyx ver. 16.0.2 software, Genetyx, Tokyo, Japan). The dendrogram was created using clustering with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Branch length indicates relative evolutionary distances. Numbers above branches are genetic distances based on 10,000 bootstrap replicates. Distances are shown as the p-distance. Subdivision groups of Arabidopsis bHLH proteins (Group IIId, IIIe, IIIf and IVa) are shown to the right of the gene names.
Figure 3.
Tomato SlTRY and SlGL3 gene expression.
(A) Real-time reverse transcription PCR analysis of SlTRY gene expression in tomato organs. (B) Real-time reverse transcription PCR analysis of SlGL3 gene expression in tomato organs. Total RNA was isolated from the indicated tissues from 7-day-old seedlings and 5-week-old plants. Expression levels of SlTRY and SlGL3 in each organ relative to those in the seedling root were shown. The experiments were repeated three times. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between the seedling root and the other organs by Student’s t-test (P<0.050).
Figure 4.
Trichome and root hair phenotypes of CPC::SlTRY transgenic plants.
(A) Trichome formation on 2-week-old Arabidopsis third leaves of wild-type Col-0 and CPC::SlTRY (#1, #2, #3, #4 and #5). (B) Trichome formation on 2-week-old Arabidopsis third leaves of wild-type Col-0, cpc-2 mutant and CPC::SlTRY in cpc-2 (#1, #2, #3, #4 and #5). Number of trichomes per leaf was determined by counting a minimum of five 2-week-old third leaves from each line. (C) Root hair formation in 5-day-old Arabidopsis seedlings of wild-type Col-0 and CPC::SlTRY (#1, #2, #3, #4 and #5). (D) Root hair formation in 5-day-old Arabidopsis seedlings of wild-type Col-0, cpc-2 mutant and CPC::SlTRY in cpc-2 (#1, #2, #3, #4 and #5). The number of root hairs per mm was determined by counting a minimum of ten 5-day-old seedlings from each line. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the transgenic lines (C), or the CPC-2 mutant and the transgenic lines (D) by Student’s t-test (P<0.050). Trichome phenotypes of wild-type Col-0 (E), cpc-2 (F), CPC::SlTRY (G) and CPC::SlTRY in cpc-2 (H). Root hair phenotypes of wild-type Col-0 (I), cpc-2 (J), CPC::SlTRY (K) and CPC::SlTRY in cpc-2 (L). Scale bars: 1 mm.
Figure 5.
Trichome and root hair phenotypes of GL3::SlGL3 transgenic plants.
(A) Trichome formation on 2-week-old Arabidopsis third leaves of wild-type Col-0 and GL3::SlGL3 (#1, #2, #3, #4 and #5). (B) Trichome formation on 2-week-old Arabidopsis third leaves of wild-type Col-0, gl3-7454 mutant and CPC::SlTRY in gl3-7454 (#1, #2, #3, #4 and #5). Number of trichomes per leaf was determined by counting a minimum of five 2-week-old third leaves from each line. (C) Root hair formation in 5-day-old Arabidopsis seedlings of wild-type Col-0 and GL3::SlGL3 (#1, #2, #3, #4 and #5). (D) Root hair formation in 5-day-old Arabidopsis seedlings of wild-type Col-0, gl3-7454 mutant and CPC::SlTRY in gl3-7454 (#1, #2, #3, #4 and #5). The number of root hairs per mm was determined by counting a minimum of ten 5-day-old seedlings from each line. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the transgenic lines [(A), (C)], or the gl3-7454 mutant and the transgenic lines [(B), (D)] by Student’s t-test (P<0.050). Trichome phenotypes of gl3-7454 (E), GL3::SlGL3 (F), and GL3::SlGL3 in gl3-7454 (G). Root hair phenotypes of gl3-7454 (H), GL3::SlGL3 (I), and GL3::SlGL3 in gl3-7454 (J). Scale bars: 1 mm.
Table 2.
Trichome branch numbers.
Figure 6.
GL2 expression in the CPC::SlTRY transgenic plants.
Real-time reverse transcription PCR analyses of the GL2 gene in wild-type Col-0 and CPC::SlTRY (#1, #2, #3, #4 and #5) (A), and wild-type Col-0, cpc-2 mutant and CPC::SlTRY in cpc-2 (#1, #2, #3, #4 and #5) (B). Expression levels were normalized to Act2 expression. An expression level of GL2 in each line relative to that in wild-type was indicated. The experiments were repeated three times. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the transgenic lines (A), or the cpc-2 mutant and the transgenic lines (B) by Student’s t-test (P<0.050).
Figure 7.
GL2 expression in CPC::SlGL3 transgenic plants.
Real-time reverse transcription PCR analyses of the GL2 gene in wild-type Col-0 and CPC::SlGL3 (#1, #2, #3, #4 and #5) (A), and wild-type Col-0, gl3-7454 mutant and CPC::SlGL3 in gl3-7454 (#1, #2, #3, #4 and #5) (B). Expression levels were normalized to Act2 expression. An expression level of GL2 in each line relative to that in wild-type was indicated. The experiments were repeated three times. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between the wild-type Col-0 and the transgenic lines (A), or the gl3-7454 mutant and the transgenic lines (B) by Student’s t-test (P<0.050).