Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and regulates root hair and trichome cell differentiation in Arabidopsis thaliana. Six additional CPC-like MYB proteins including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ENHANCER OF TRY AND CPC2 (ETC2), ENHANCER OF TRY AND CPC3/CPC-LIKE MYB3 (ETC3/CPL3), TRICHOMELESS1 (TCL1), and TRICHOMELESS2/CPC-LIKE MYB4 (TCL2/CPL4) also have the ability to regulate root hair and/or trichome cell differentiation in Arabidopsis. In this review, we describe our latest findings on how CPC-like MYB transcription factors regulate root hair cell differentiation. Recently, we identified the tomato SlTRY gene as an ortholog of the Arabidopsis TRY gene. Transgenic Arabidopsis plants harboring SlTRY produced more root hairs, a phenotype similar to that of 35S::CPC transgenic plants. CPC is also known to be involved in anthocyanin biosynthesis. Anthocyanin accumulation was repressed in the SlTRY transgenic plants, suggesting that SlTRY can also influence anthocyanin biosynthesis. We concluded that tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and that a CPC-like R3 MYB may be a key common regulator of plant root-hair development.


BRIEF BACKGROUND
Cell fate determination is a critical step in plant development. In growing roots, epidermal cells differentiate into two cell types, root-hair cells, and non-hair cells in a file-specific manner. In Arabidopsis roots, epidermal cells in eight symmetrically positioned files differentiate into root-hair cells, and the cells of the other files become non-hair cells. Morphological analysis has shown the positional relationship between cortical cells and epidermal cells. Epidermal cells in contact with the junction of two underlying cortical cells differentiate into root-hair cells, whereas the cells in contact with only one cortical cell differentiate into non-hair cells (Dolan et al., 1993(Dolan et al., , 1994Galway et al., 1994;Berger et al., 1998). Several regulatory factors are involved in root-hair or non-hair cell differentiation. The glabra 2 (gl2) and werewolf (wer) mutants convert non-hair cells to root hair cells (Masucci et al., 1996;Lee and Schiefelbein, 1999). The GL2 gene encodes a homeodomain leucine-zipper protein, and the WER gene encodes an R2R3-type MYB transcription factor that activates GL2 expression preferentially in differentiating non-hair cells (Rerie et al., 1994;Di Cristina et al., 1996;Masucci et al., 1996;Lee and Schiefelbein, 1999). GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) encode basic helixloop-helix (bHLH) transcription factors that affect non-hair cell differentiation in a redundant manner, as evidenced by the conversion of non-hair cells to root-hair cells in the gl3 egl3 double mutant (Bernhardt et al., 2003). Although, obvious increase in the number of root-hair cells was hardly observed in both gl3 and egl3 single mutants (Bernhardt et al., 2003). The TRANSPAR-ENT TESTA GLABRA1 (TTG1) gene is also involved in non-hair cell fate determination, as shown by the conversion of non-hair cells to root-hair cells in the ttg1 mutant (Galway et al., 1994). The TTG1 gene encodes a WD40-repeat protein (Walker et al., 1999). GL3 and EGL3 interact with WER (Bernhardt et al., 2003) and with TTG1 (Payne et al., 2000;Esch et al., 2003;Zhang et al., 2003) in yeast cells. A protein complex including WER, GL3/EGL3, and TTG1 acts upstream of the GL2 gene in the root-hair regulatory pathway and promotes GL2 gene expression (Galway et al., 1994;Rerie et al., 1994;Wada et al., 1997;Hung et al., 1998;Lee and Schiefelbein, 1999;Bernhardt et al., 2003Bernhardt et al., , 2005. The cells expressing GL2 differentiate into non-hair cells (Figure 1). In contrast, the root-hair cell differentiation is controlled by CAPRICE (CPC) as shown by a few root-hair phenotype of the cpc mutant (Wada et al., 1997). The CPC gene encodes R3-type MYB protein (Wada et al., 1997). The TTG1-GL3/EGL3-WER protein complex also up-regulates CPC gene expression in non-hair cells (Koshino-Kimura et al., 2005). The CPC protein moves from non-hair cells to neighboring cells and disturbs the formation of the TTG1-GL3/ETC3-WER transcriptional complex by competitively binding with WER Koshino-Kimura et al., 2005;Kurata et al., 2005;Tominaga et al., 2007). The formation of the TTG1-GL3/EGL3-CPC protein complex represses expression of GL2, thereby inhibiting non-hair cell differentiation Kurata et al., 2005; Figure 1).

THE CPC FAMILY PROMOTES ROOT-HAIR CELL DIFFERENTIATION
CAPRICE encodes a small protein with an R3 MYB motif and strongly promotes root-hair cell differentiation in Arabidopsis (Wada et al., 1997). In addition, we presented a model in which www.frontiersin.org FIGURE 1 | Regulation of root-hair cell and non-hair cell fate determination by transcription factors. The TTG1-GL3/EGL3-WER complex promotes GL2 and CPC expression. The cell expressing GL2 differentiates into a non-hair cell. The CPC protein moves from the non-hair cell to a neighboring cell and competes with WER for binding to GL3/EGL3. The TTG1-GL3/EGL3-CPC complex cannot promote GL2 expression, resulting in the differentiation of a root-hair cell.

RECENT FINDINGS ON THE FUNCTIONS OF THE CPC FAMILY
We have identified the CPL4 gene between At2g30430 and ETC2 (At2g30420) independently of Gan et al. (Gan et al., 2011;Tominaga-Wada and Nukumizu, 2012). Between CPL4 and ETC2, there were several chimeric transcripts generated through alternative splicing (Tominaga-Wada and Nukumizu, 2012). Our study proposed that inter-genic alterative splicing also characterizes the CPC-like MYB gene family (Tominaga-Wada and Nukumizu, 2012).
A lateral inhibition mechanism mediated by cell-to-cell movement of CPC was thought to cause cell fate specification (Lee and Schiefelbein, 2002;Schiefelbein, 2007, 2008). However, it is unclear how CPC, which is preferentially expressed in non-hair cells, specifically acts in the root-hair cells rather than in non-hair cells. Recently, nuclear trapping of CPC in the roothair cells by EGL3 was suggested to be involved in root-hair cell differentiation (Kang et al., 2013). CPC protein accumulates predominantly in the nuclei of root-hair cells in the early meristematic region, and this localization requires specific expression of EGL3 in the root-hair cells (Kang et al., 2013). These results suggest that cell-to-cell movement of CPC occurs within the meristem of root epidermal cells and that EGL3 traps the CPC protein in the root-hair cells (Kang et al., 2013). CPC and TRY were reported to recruit AtMYC1 into the nucleus, suggesting mutual control of the intracellular localization of patterning proteins (Pesch et al., 2013). AtMYC1, a homologue of GL3 and EGL3, encodes a bHLH transcription factor predominantly localized in the cytoplasm (Urao et al., 1996;Pesch et al., 2013). AtMYC1 regulates the distribution of GL1 protein between the nucleus and the cytoplasm. On the other hand, AtMYC1 is recruited into the nucleus by TRY and CPC, subsequent to significant accumulation of TRY and CPC in the nucleus (Pesch et al., 2013). These results and genetic analyses imply that AtMYC1 represses the activity of TRY and CPC (Pesch et al., 2013).
Tissue-specific transcript profiling also indicated that there were some redundancies between CPC and TRY at the transcriptional level (Simon et al., 2013). We have extended the characterization of CPC-like MYB genes to include the identification of inter-genic alterative splicing and precise expression patterns using tissue-specific transcript profiling (Tominaga-Wada and Nukumizu, 2012;Simon et al., 2013). Recent findings have also revealed that in addition to the formation of the transcription complex, each type of transcription factor can regulate the inter-and intra-cellular localization of the other types to regulate root hair and trichome formation (Kang et al., 2013;Pesch et al., 2013).

A CPC -LIKE MYB IN TOMATO
Recently, we identified the tomato SlTRY gene as an ortholog of an Arabidopsis CPC-like MYB gene (Tominaga-Wada et al., 2013b). The CPC::SlTRY construct in cpc-2 transgenic plants increased the number of root-hairs compared with that of the cpc-2 mutant plants (Figure 2; Tominaga-Wada et al., 2013b). These results suggest that tomato and Arabidopsis use common transcription factors for root-hair differentiation. In addition to root-hair cell differentiation, the Arabidopsis CPC gene is known to regulate anthocyanin biosynthesis (Zhu et al., 2009). Anthocyanin accumulation was repressed in the CPC::SlTRY transgenic plants as was observed in the 35S::CPC transgenic plants, suggesting that SlTRY also influences anthocyanin pigment synthesis (Tominaga-Wada et al., 2013a). Tomato and Arabidopsis partially use similar transcription factors for root hair cell differentiation, and a CPC-like R3 MYB may be a key common regulator of plant root-hair development. Further analysis of CPC-like gene function in tomato is on-going.

FUTURE PERSPECTIVES
The cell-to-cell movement of CPC from non-hair cells to roothair cells is important for root-hair cell specification; however, the precise mechanism of CPC movement is unknown. How CPC is targeted, transported through plasmodesmata, and trapped in the nucleus of the root-hair cells to define cell fate is an intriguing problem.
Transcriptome analyses provide detailed characterizations of transcription factors involved in root epidermal cell differentiation. Further molecular characterization of individual genes and mutant phenotypes is necessary to fully assess the precise mechanism for root epidermal cell differentiation, including an analysis of redundancies in the epidermal cell regulatory pathway.
TRY and GL3 homologous genes were identified from the tomato genome and named SlTRY and SlGL3, respectively (Tominaga-Wada et al., 2013b). SlTRY showed a similar function to TRY, including inhibition of trichome formation and enhancement of root-hair differentiation. On the other hand, SlGL3 did not show any obvious effect on trichome or nonhair cell differentiation (Tominaga-Wada et al., 2013b). There may be other GL3 ortholog(s) in the unannotated tomato genomes, or tomato uses other pathways to regulate epidermal cell differentiation. Further studies to determine the functions of R3-MYB and bHLH in epidermal cell differentiation in tomato are required.

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