TRICHOMELESS1 Research Articles

The TCP4 Transcription Factor Directly Activates TRICHOMELESS1 and 2 and Suppresses Trichome Initiation.

Trichomes are the first line of defense on the outer surface of plants against biotic and abiotic stresses. Because trichomes on leaf surfaces originate from the common epidermal progenitor cells that also give rise to pavement cells and stomata, their density and distribution are under strict genetic control. Regulators of trichome initiation have been identified and incorporated into a biochemical pathway wherein an initiator complex promotes trichome fate in an epidermal progenitor cell, while an inhibitor complex suppresses it in the neighboring cells. However, it is unclear how these regulator proteins, especially the negative regulators, are induced by upstream transcription factors and integrated with leaf morphogenesis. Here, we show that the Arabidopsis (Arabidopsis thaliana) class II TCP proteins activate TRICHOMELESS1 (TCL1) and TCL2, the two established negative regulators of trichome initiation, and reduce trichome density on leaves. Loss-of-function of these TCP proteins increased trichome density whereas TCP4 gain-of-function reduced trichome number. TCP4 binds to the upstream regulatory elements of both TCL1 and TCL 2 and directly promotes their transcription. Further, the TCP-induced trichome suppression is independent of the SQUAMOSA PROMOTER BINDING PROTEIN LIKE family of transcription factors, proteins that also reduce trichome density at later stages of plant development. Our work demonstrates that the class II TCP proteins couple leaf morphogenesis with epidermal cell fate determination.

Single-repeat R3 MYB transcription factors from Platanus acerifolia negatively regulate trichome formation in Arabidopsis.

Four R3 MYB genes were cloned and identified from Platanus acerifolia and analysed according to endogenous gene expression profiles, protein-protein interaction patterns, phenotypic effects and related gene expression profiles in transgenic Arabidopsis, suggesting that London plane R3 MYB genes inhibit trichome formation in Arabidopsis. The CPC-like MYB transcription factors including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC 1, 2 and 3 (ETC1, ETC2 and ETC3), TRICHOMELESS1 (TCL1) and TRICHOMELESS2(TCL2) play important roles in controlling trichome patterning in Arabidopsis. In this study, four sequences homologous with the Arabidopsis CPC family were identified from London plane and named PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3. Over-expression of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 in Arabidopsis resulted in glabrous phenotypes. In addition, expression of endogenous GL2, GL1, MYB23, TTG2 and a set of R3 MYB-encoding genes was markedly reduced. Furthermore, the protein products of PaTRY, PaCPC-like1, PaCPC-like2 and PaCPC-like3 were shown to interact with PaGL3 in yeast two-hybrid assays. Together, these results likely suggest that the mechanisms of trichome regulation in London plane have similarities with those in Arabidopsis.

Identification and Functional Characterization of R3 MYB Transcription Factor Genes in Soybean

In Arabidopsis, trichome formation is regulated by a MYB-bHLH-WD40 (MBW) transcriptional activator complex, which can activate the expression of GLABRA2 (GL2) and R3 MYB genes. GL2 is required for trichome formation, whereas R3 MYBs inhibit trichome formation by blocking the formation of the MBW complex, thus inhibiting the expression of GL2. By using the amino acid sequence of the Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1) to BLAST the soybean (Glycine max) protein database, we found that there are a total of six R3 MYB genes in soybean, namely Glycine max TRICHOMELESS1 through 6 (GmTCL1-GmTCL6). By generating transgenic plants, we found that trichome formation in soybean plants overexpressing each of the GmTCLs remained largely unchanged, and the expression of putative GL1 and GL2 genes in the transgenic plants was unaffected. However, all the GmTCLs interacted with GLABRA3 (GL3) in transfected Arabidopsis protoplasts, expression each of the GmTCLs in Arabidopsis inhibited trichome formation, and the expression levels of GL1 and GL2 were greatly reduced in the Arabidopsis transgenic plants. Moreover, phenotypic complementary analysis showed that GmTCL1 is functionally equivalent to TCL1. Taken together, these results suggest that GmTCLs are functional R3 MYBs, however, they do not regulate trichome formation in soybean.

NTL8 Regulates Trichome Formation in Arabidopsis by Directly Activating R3 MYB Genes TRY and TCL1.

The NAM, ATAF1/2, and CUC (NAC) are plant-specific transcription factors that regulate multiple aspects of plant growth and development and plant response to environmental stimuli. We report here the identification of NTM1-LIKE8 (NTL8), a membrane-associated NAC transcription factor, as a novel regulator of trichome formation in Arabidopsis (Arabidopsis thaliana). From an activation-tagged Arabidopsis population, we identified a dominant, gain-of-function mutant with glabrous inflorescence stem. By using plasmid rescue and RT-PCR analyses, we found that NTL8 was tagged; thus, the mutant was named ntl8-1 Dominant (ntl8-1D). Recapitulation experiment further confirmed that the phenotype observed in the ntl8-1D mutant was caused by elevated expression of NTL8 Quantitative RT-PCR results showed that the expression level of the single-repeat R3 MYB genes TRIPTYCHON (TRY) and TRICHOMELESS1 (TCL1) was elevated in the ntl8-1D mutant. Genetic analyses demonstrated that NTL8 acts upstream of TRY and TCL1 in the regulation of trichome formation. When recruited to the promoter region of the reporter gene Gal4:GUS by a fused GAL4 DNA-binding domain, NTL8 activated the expression of the reporter gene. Chromatin immunoprecipitation results indicated that TRY and TCL1 are direct targets of NTL8. However, NTL8 did not interact with SQUAMOSA PROMOTER BINDING PROTEIN LIKE9, another transcription factor that regulates the expression of TRY and TCL1, in yeast and plant cells. Taken together, our results suggest that NTL8 negatively regulates trichome formation in Arabidopsis by directly activating the expression of TRY and TCL1.

Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation.

In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation.

Control of trichome formation in Arabidopsis by poplar single-repeat R3 MYB transcription factors.

In Arabidopsis, trichome formation is regulated by the interplay of R3 MYBs and several others transcription factors including the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), the R2R3 MYB transcription factor GLABRA1 (GL1), the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3), and the homeodomain protein GLABRA2 (GL2). R3 MYBs including TRICHOMELESS1 (TCL1), TCL2, TRYPTICHON (TRY), CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and ETC3 negatively regulate trichome formation by competing with GL1 for binding GL3 or EGL3, thus blocking the formation of TTG1–GL3/EGL3–GL1, an activator complex required for the activation of the trichome positive regulator gene GL2. However, it is largely unknown if R3 MYBs in other plant species especially woody plants have similar functions. By BLASTing the Populus trichocarpa protein database using the entire amino acid sequence of TCL1, an Arabidopsis R3 MYB transcription factor, we identified a total of eight R3 MYB transcription factor genes in poplar, namely P. trichocarpa TRICHOMELESS1 through 8 (PtrTCL1–PtrTCL8). The amino acid signature required for interacting with bHLH transcription factors and the amino acids required for cell-to-cell movement of R3 MYBs are not fully conserved in all PtrTCLs. When tested in Arabidopsis protoplasts, however, all PtrTCLs interacted with GL3. Expressing each of the eight PtrTCL genes in Arabidopsis resulted in either glabrous phenotypes or plants with reduced trichome numbers, and expression levels of GL2 in all transgenic plants tested were greatly reduced. Expression of PtrTCL1 under the control of TCL1 native promoter almost completely complemented the mutant phenotype of tcl. In contrast, expression of PtrTCL1 under the control of TRY native promoter in the try mutant, or under the control of CPC native promoter in the cpc mutant resulted in glabrous phenotypes, suggesting that PtrTCL1 functions similarly to TCL1, but not TRY and CPC.

Interaction between Two Timing MicroRNAs Controls Trichome Distribution in Arabidopsis

The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors function as an endogenous age cue in regulating plant phase transition and phase-dependent morphogenesis, but the control of SPL output remains poorly understood. In Arabidopsis thaliana the spatial pattern of trichome is a hallmark of phase transition and governed by SPLs. Here, by dissecting the regulatory network controlling trichome formation on stem, we show that the miR171-targeted LOST MERISTEMS 1 (LOM1), LOM2 and LOM3, encoding GRAS family members previously known to maintain meristem cell polarity, are involved in regulating the SPL activity. Reduced LOM abundance by overexpression of miR171 led to decreased trichome density on stems and floral organs, and conversely, constitutive expression of the miR171-resistant LOM (rLOM) genes promoted trichome production, indicating that LOMs enhance trichome initiation at reproductive stage. Genetic analysis demonstrated LOMs shaping trichome distribution is dependent on SPLs, which positively regulate trichome repressor genes TRICHOMELESS 1 (TCL1) and TRIPTYCHON (TRY). Physical interaction between the N-terminus of LOMs and SPLs underpins the repression of SPL activity. Importantly, other growth and developmental events, such as flowering, are also modulated by LOM-SPL interaction, indicating a broad effect of the LOM-SPL interplay. Furthermore, we provide evidence that MIR171 gene expression is regulated by its targeted LOMs, forming a homeostatic feedback loop. Our data uncover an antagonistic interplay between the two timing miRNAs in controlling plant growth, phase transition and morphogenesis through direct interaction of their targets.

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.

Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYB transcription factor in the regulation of trichome patterning in Arabidopsis

BackgroundSingle-repeat R3 MYB transcription factors (single-repeat MYBs) play important roles in controlling trichome patterning in Arabidopsis. It was proposed that single-repeat MYBs negatively regulate trichome formation by competing with GLABRA1 (GL1) for binding GLABRA3/ENHANCER OF GLABRA3 (GL3/EGL3), thus inhibiting the formation of activator complex TTG1(TRANSPARENT TESTA GLABRA1)-GL3/EGL3-GL1 that is required for the activation of GLABRA2 (GL2), whose product is a positive regulator of trichome formation. Previously we identified a novel single-repeat MYB transcription factor, TRICHOMELESS1 (TCL1), which negatively regulates trichome formation on the inflorescence stems and pedicels by directly suppressing the expression of GL1.ResultsWe analyzed here the role of TRICHOMELESS2 (TCL2), a previously-uncharacterized single-repeat MYB transcription factor in trichome patterning in Arabidopsis. We showed that TCL2 is closely related to TCL1, and like TCL1 and other single-repeat MYBs, TCL2 interacts with GL3. Overexpression of TCL2 conferred glabrous phenotype while knockdown of TCL2 via RNAi induced ectopic trichome formation on the inflorescence stems and pedicels, a phenotype that was previously observed in tcl1 mutants. These results suggested that TCL2 may have overlapping function with TCL1 in controlling trichome formation on inflorescences. On the other hand, although the transcription of TCL2, like TCL1, is not controlled by the activator complex formed by GL1 and GL3, and TCL2 and TCL1 proteins are more than 80% identical at the amino acid level, the expression of TCL2 under the control of TCL1 promoter only partially recovered the mutant phenotype of tcl1, implying that TCL2 and TCL1 are not fully functional equivalent.ConclusionsTCL2 function redundantly with TCL1 in controlling trichome formation on inflorescences, but they are not fully functional equivalent. Transcription of TCL2 is not controlled by activator complex formed by GL1 and GL3, but MIR156 controlled SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) transcription factors. However, SPLs might require co-activators to regulate the expression of their target genes, including TCL1, TRY and possibly, TCL2.

Temporal Control of Trichome Distribution by MicroRNA156-TargetedSPLGenes inArabidopsis thaliana

The production and distribution of plant trichomes is temporally and spatially regulated. After entering into the flowering stage, Arabidopsis thaliana plants have progressively reduced numbers of trichomes on the inflorescence stem, and the floral organs are nearly glabrous. We show here that SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes, which define an endogenous flowering pathway and are targeted by microRNA 156 (miR156), temporally control the trichome distribution during flowering. Plants overexpressing miR156 developed ectopic trichomes on the stem and floral organs. By contrast, plants with elevated levels of SPLs produced fewer trichomes. During plant development, the increase in SPL transcript levels is coordinated with the gradual loss of trichome cells on the stem. The MYB transcription factor genes TRICHOMELESS1 (TCL1) and TRIPTYCHON (TRY) are negative regulators of trichome development. We show that SPL9 directly activates TCL1 and TRY expression through binding to their promoters and that this activation is independent of GLABROUS1 (GL1). The phytohormones cytokinin and gibberellin were reported to induce trichome formation on the stem and inflorescence via the C2H2 transcription factors GIS, GIS2, and ZFP8, which promote GL1 expression. We show that the GIS-dependent pathway does not affect the regulation of TCL1 and TRY by miR156-targeted SPLs, represented by SPL9. These results demonstrate that the miR156-regulated SPLs establish a direct link between developmental programming and trichome distribution.

Comprehensive analysis of single-repeat R3 MYB proteins in epidermal cell patterning and their transcriptional regulation in Arabidopsis

BackgroundSingle-repeat R3 MYB transcription factors are critical components of the lateral inhibition machinery that mediates epidermal cell patterning in plants. Sequence analysis of the Arabidopsis genome using the BLAST program reveals that there are a total of six genes, including TRIPTYCHON (TRY), CAPRICE (CPC), TRICHOMELESS1 (TCL1), and ENHANCER of TRY and CPC 1, 2, and 3 (ETC1, ETC2 and ETC3) encoding single-repeat R3 MYB transcription factors that are approximately 50% identical to one another at the amino acid level. Previous studies indicate that these single-repeat R3 MYBs regulate epidermal cell patterning. However, each of the previous studies of these single-repeat R3 MYBs has been limited to an analysis of only a subset of these six genes, and furthermore, they have limited their attention to epidermal development in only one or two of the organs. In addition, the transcriptional regulation of these single-repeat R3 MYB genes remains largely unknown.ResultsBy analyzing multiple mutant lines, we report here that TCL1 functions redundantly with other single-repeat R3 MYB transcription factors to control both leaf trichome and root hair formation. On the other hand, ETC1 and ETC3 participate in controlling trichome formation on inflorescence stems and pedicles. Further, we discovered that single-repeat R3 MYBs suppress trichome formation on cotyledons and siliques, organs that normally do not bear any trichomes. By using Arabidopsis protoplast transfection assays, we found that all single-repeat R3 MYBs examined interact with GL3, and that GL1 or WER and GL3 or EGL3 are required and sufficient to activate the transcription of TRY, CPC, ETC1 and ETC3, but not TCL1 and ETC2. Furthermore, only ETC1's transcription was greatly reduced in the gl3 egl3 double mutants.ConclusionOur comprehensive analysis enables us to draw broader conclusions about the role of single-repeat R3 MYB gene family than were possible in the earlier studies, and reveals the genetic basis of organ-specific control of trichome formation. Our findings imply the presence of multiple mechanisms regulating the transcription of single-repeat R3 MYB genes, and provide new insight into the lateral inhibition mechanism that mediates epidermal cell patterning.

TRICHOMELESS1 regulates trichome patterning by suppressingGLABRA1inArabidopsis

The patterning of epidermal cell types in Arabidopsis is a simple and useful model for studying the molecular basis of cell specification in plants. The distribution of different cell types in the Arabidopsis epidermis is regulated by a lateral inhibition mechanism that relies on interactions between transcription factors. However, it is unclear how temporal- or organ-specific differences in epidermal patterning are achieved. Here we identify TRICHOMELESS1 (TCL1) as a new and major single-repeat MYB-type transcription factor that negatively regulates trichome formation in the inflorescence epidermis. A dominant mutant with elevated expression of TCL1 has a glabrous (trichomeless) phenotype, whereas a loss-of-function mutation in TCL1 uniquely confers ectopic trichome formation on inflorescence stem and pedicels. Genetic analyses demonstrate that TCL1 and CAPRICE work synergistically to regulate trichome patterning on these organs. Interestingly, overexpression of TCL1 specifically suppresses the expression of GLABRA1 (GL1), a crucial component in the trichome initiation complex, whereas loss-of-function of TCL1 enhances GL1 expression. Chromatin immunoprecipitation results show that TCL1 can be recruited to the cis-acting regulatory elements of GL1. These results provide the first molecular and genetic evidence that an R3 MYB may negatively regulate trichome cell specification in a novel manner by directly suppressing the transcription of GL1.