Expression Analysis of an R3-Type MYB Transcription Factor CPC-LIKE MYB4 (TRICHOMELESS2) and CPL4-Related Transcripts in Arabidopsis

The CAPRICE (CPC)-like MYB gene family encodes R3-type MYB transcription factors in Arabidopsis. There are six additional CPC-like MYB sequences in the Arabidopsis genome, including TRYPTICHON (TRY), ENHANCER OF TRY AND CPC1 and 2 (ETC1 and ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), and TRICHOMELESS1 and 2 (TCL1 and TCL2). We independently identified CPC-LIKE MYB4 (CPL4), which was found to be identical to TCL2. RT-PCR analysis showed that CPL4 is strongly expressed in shoots, including true leaves, but not in roots. Promoter-GUS analyses indicated that CPL4 is specifically expressed in leaf blades. Although CPC expression was repressed in 35S::ETC1, 35S::ETC2 and 35S::CPL3 backgrounds, CPL4 expression was not affected by ETC1, ETC2 or CPL3 over-expression. Notably, several chimeric transcripts may result from inter-genic alternative splicing of CPL4 and ETC2, two tandemly repeated genes on chromosome II. At least two chimeric transcripts named CPL4-α and CPL4-β are expected to encode complete CPC-like MYB proteins

Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis

<p>The <i>CAPRICE</i> (<i>CPC</i>) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in <i>Arabidopsis</i>. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The <i>ENHANCER OF TRY AND CPC1</i> (<i>ETC1</i>) gene encodes the <i>CPC</i> homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, <i>CPCp:S1:ETC1:2xGFP</i> was constructed and introduced into <i>Arabidopsis</i>. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.</p

Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) Control Tomato (<i>Solanum lycopersicum</i>) Anthocyanin Biosynthesis

<div><p>In <i>Arabidopsis thaliana</i> the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes <i>SlTRY</i> (<i>CPC</i>) and <i>SlGL3</i> (<i>GL3</i>) into <i>Arabidopsis</i>, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and <i>Arabidopsis</i> partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, <i>35S::CPC</i> inhibited anthocyanin accumulation, whereas <i>GL3::GL3</i> enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including <i>Phe-ammonia lyase</i> (<i>PAL</i>), the flavonoid pathway genes <i>chalcone synthase</i> (<i>CHS</i>), <i>dihydroflavonol reductase</i> (<i>DFR</i>), and <i>anthocyanidin synthase</i> (<i>ANS</i>) were repressed in <i>35S::CPC</i> tomato. In contrast, the expression levels of <i>PAL</i>, <i>CHS</i>, <i>DFR</i>, and <i>ANS</i> were significantly higher in <i>GL3::GL3</i> tomato compared with control plants. These results suggest that <i>CPC</i> and <i>GL3</i> also influence anthocyanin pigment synthesis in tomato.</p></div

Phenotypes of <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato plants.

<p>(A) Two-week old control plant. (B) Adaxial side of the first true leaf from the plant shown in A. (C) Abaxal side of the first true leaf from the plant shown in A. (D) Two-week-old <i>35S::CPC</i> transgenic plant. (E) Adaxial side of the first true leaf from the plant shown in D. (F) Abaxal side of the first true leaf from the plant shown in D. (G) Two-week-old <i>GL3::GL3</i> transgenic plant. (H) Adaxial side of the first true leaf from the plant shown in G. (I) Abaxal side of the first true leaf from the plant shown in G. Scale bars: 1 cm in A for A, D and G; 5 mm in B for B, C, E, F, H and I.</p

Leaf and root epidermal phenotypes of <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato plants.

<p>(A) The first true leaf from the two-week old control plant. (B) Close-up view of the adaxial side of the leaf shown in A. (C) Five-day-old seedling roots of control plants. (D) The first true leaf from the two-week old <i>35S::CPC</i> plant. (E) Close-up view of the adaxial side of the leaf shown in E. (F) Five-day-old seedling roots of <i>35S::CPC</i> plants. (G) The first true leaf from the two-week old <i>GL3::GL3</i>plant. (H) Close-up view of the adaxial side of the leaf shown in G. (I) Five-day-old seedling roots of <i>GL3::GL3</i> plants. Scale bars: 1 mm in A, C, D, F, G and I; 20 µm in B, E and H.</p

Amino acid substitutions in CPC-LIKE MYB reveal residues important for protein stability in Arabidopsis roots.

TRYPTICHON (TRY) and ENHANCER OF TRY AND CPC2 (ETC2) encode R3-type MYB transcription factors that are involved in epidermal cell differentiation in Arabidopsis thaliana. TRY and ETC2 belong to the CPC-like MYB gene family, which includes seven homolog genes. Previously, we showed that among the CPC family members, TRY and ETC2 are characterized by rapid proteolysis compared with that of other members, and we demonstrated that this proteolysis is mediated by the proteasome-dependent pathway. In this study, we compared the functions of the wild-type TRY and ETC2 proteins and their amino acid-substituted versions. Our results showed that the substitution of amino acids in the C-terminal of TRY and ETC2 conferred them the ability to induce root hair formation. Furthermore, we confirmed that these mutations enhanced the stability of the TRY and ETC2 proteins. These results revealed that the amino acids, which are important for the functions of TRY and ETC2, mediate morphological pattern formation and can be useful in understanding the pathway determining the fate of root hair cells

Expression analysis of genes associated with the anthocyanin biosynthetic pathway in tomato leaves.

<p>Enzyme names of the anthocyanin biosynthetic pathway are abbreviated as follows: phenyl alanine ammonia-lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (ANS). (A) Real-time reverse transcription PCR analysis of <i>PAL</i> gene expression in <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato leaves. (B) Real-time reverse transcription PCR analysis of <i>CHS</i> gene expression in <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato leaves. (C) Real-time reverse transcription PCR analysis of <i>DFR</i> gene expression in <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato leaves. (D) Real-time reverse transcription analysis of <i>ANS</i> gene expression in <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato leaves. Total RNA was isolated from the indicated leaves from two-week-old tomato plants. Expression levels of <i>PAL</i>, <i>CHS</i>, <i>DFR and ANS3</i> in each sample relative to those in the control plants are shown. The experiments were repeated three times. Error bars indicate the standard error. Bars marked with asterisks indicate a significant difference between control and indicated transgenic plants by Student's <i>t</i>-test (P<0.050).</p

Stem phenotypes of <i>35S::CPC</i> and <i>GL3::GL3</i> transgenic tomato plants.

<p>(A) Transverse section of a hypocotyl of a two-week-old control plant. (B) Transverse section of a hypocotyl of a two-week-old <i>35S::CPC</i> transgenic plant. (C) Transverse section of a hypocotyl of a two-week-old <i>GL3::GL3</i> transgenic plant. Scale bars: 100 µm.</p