TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex

Abstract

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Figure 1

CIN-like TCP transcription factors participate in repressing cotyledon trichome formation. A, Six-day-old seedlings of wild-type and tcp2/3/4/5/10/13/17 mutant plants. The red boxes indicate close-up views of the cotyledons. The ectopic trichomes are clearly produced on cotyledons of tcp2/3/4/5/10/13/17 mutants. Bars = 1 mm. B–D, Images of the 6-d-old seedlings of the TCP4pro-TCP4-Flag (C) or TCP10pro-TCP10-Flag (D) lines showing that TCP4 or TCP10 complements the ectopic trichomes on cotyledons of tcp2/3/4/5/10/13/17 (B). Bars = 1 mm. E–J, Scanning electronic micrographs of the cotyledon trichome cells of 3-, 4-, and 6-d-old wild-type plants (E–G) and tcp2/3/4/5/10/13/17 mutants (H–J). The red arrows indicate the initiated trichome stalks on the 4-d-old tcp2/3/4/5/10/13/17 cotyledons I, Bars = 50 μm. K–M, CIN-like TCP transcription factors have redundant and different contributions to inhibiting trichome formation on cotyledons. The tcp2/3/4/5/10/13 sextuple mutant (M) produced fewer trichomes (red arrow) on cotyledons than the tcp2/3/4/5/10/13/17 septuple mutant, while the tcp2/3/4/5/13/17 (K) and tcp3/4/5/10/13/17 (L) sextuple mutants displayed no trichomes on cotyledons. Bars = 1 mm.

Figure 2

TCPs interacted with the bHLH transcription factor GL3. A, Schematic diagrams of TCP4 protein and truncated forms. The black rectangles represent the TCP domain. B, The yeast-two-hybrid assays of GL3, EGL3, and TCP4 proteins. The transformed yeasts were spotted on selection medium (SD–Leu–Trp–His) containing 5 mM 3-AT at dilutions of 10-, 100-, and 1,000-fold. The TCP4ΔN had weak self-activation activity in yeast, so 5 mM 3-AT (3-amino-1, 2, 4-triazole) was used to inhibit the self-activation for the cotransformed yeasts using TCP4ΔN as the bait. The selection medium used for TCP4ΔC and EGL3 as baits did not contain 3-AT. AD, activation domain; DBD, DNA-binding domain. C, Firefly luciferase complementation assay to test the interaction between TCP4 and GL3 in N. benthamiana. The miR319-resistant TCP4 protein was fused with nLUC at its C-terminus and GL3 protein was fused with cLUC at its N-terminus. nLUC and cLUC fused with GAL4BD were used as negative controls. D, Co-IP assay to confirm the interaction between TCP4 and GL3 in vivo. E, Schematic diagrams of GL3 protein and truncated forms. The black rectangles in the schematic diagrams of GL3 represent the bHLH domain. F, Yeast-two-hybrid assays of truncated GL3 and TCP4. The selection medium contained 5 mM 3-AT. G, Yeast-two-hybrid assays of GL3 and CIN-like TCPs (including TCP2, TCP3, TCP4, TCP5, TCP10, TCP13, TCP17, and TCP24). GL3 was fused with the DNA-binding domain (DBD) as bait. The transformed yeasts were spotted on selection medium (SD–Leu–Trp–His) containing 5 mM 3-AT at dilutions of 10-, 100-, and 1,000-fold.

Figure 3

TCP4 and the members of the MBW complex are expressed ubiquitously in cotyledons after germination. A, Relative expression levels of GL1, GL3, TTG1, and EGL3 in 3- and 6-d-old wild-type cotyledons. The expression levels of corresponding genes in the fifth true leaves of 21-d-old wild-type plants were set as 1. ACTIN8 was used as the reference gene. The data are the mean (±SE) of three biological replicates. B–K, Expression patterns of GL3 and TCP4 in 3- to 6-d-old wild-type cotyledons with GUS histochemical staining. Bars = 200 μm. B and C, Expression pattern of GL3 (B) and TCP4 (C) in 3-d-old cotyledons. D and E, Expression pattern of GL3 (D) and of TCP4 (E) in 4-d-old cotyledons. F and G, Expression pattern of GL3 (F) and TCP4 (G) in 5-d-old cotyledons. H and I, Expression pattern of GL3 (H) and TCP4 (I) in 6-d-old cotyledons. J and K, Expression pattern of GL3 (J) and TCP4 (K) in 7-d-old cotyledons.

Figure 4

TCP proteins disrupt the function of the MBW complex in repressing trichome formation on cotyledons. A, Firefly luciferase complementation assay showed that TCP4 interfered with the interaction between GL1 and GL3 in Arabidopsis protoplasts. The combinations are listed at the upper right corner. The combinations including GAL4BD-nluc and cluc-GL1, GL3-nluc and cluc-GAL4BD, and GAL4BD-nluc and cluc-GAL4BD were used as negative controls. The data are the means (±se) of three biological replicates. B, The transient expression assay showed that the activation of GL2 expression by GL1/GL3 was repressed by TCP4. The schematic diagram shows the constructs used in the transient expression assays. The GL2pro-LUC reporter was cotransformed with corresponding constructs. The data are the means (±se) of four biological replicates. The asterisks represent Student’s t test significance compared with GL1+GL3. ***P <0.001. C, The relative expression levels of GL2 in the 3- to 8-d-old cotyledons of wild-type and tcp2/3/4/5/10/13/17 plants. The expression level of GL2 in 3-d-old wild-type cotyledons was set as one. ACTIN8 was used as the reference gene. The data are the means (±se) of three biological replicates. The asterisks represent Student’s t test significance. **P < 0.01; ***P < 0.001. D–F, The genetic complementation of ectopic trichome on cotyledons of tcp2/3/4/5/10/13/17 mutants by loss-of-function of GL1 or GL3. Cotyledons of 6-d-old gl1 tcp2/3/4/5/10/13/17 (E) and gl3 tcp2/3/4/5/10/13/17 (F) mutants were glabrous. White arrows indicate the trichomes on tcp2/3/4/510/13/17 cotyledons (D). Bars = 500 μm.

Figure 5

TCPs directly promote the expression levels of R3 MYB genes. A, Peak graphs showing the ChIP-seq raw reads at the potential TCP4-bound gene loci using 4-d-old dark-grown wild-type and 35S-Myc-mTCP4 seedlings. The black bars indicate the genomic regions of the genes. The blue arrows indicate the transcriptional directions. Thin bars = 500 bp. B, Schematic diagrams of the upstream regions of CPC, ETC1, ETC3, and TCL2. The vertical lines indicate the potential TCP4-binding motif GGACCA. The black arrows represent the transcriptional start sites. CPC-I: −2,675 to –2,670 bp; CPC-II: −746 to −741 bp; CPC-III: −736 to −731 bp; ETC1-I: −923 to −918 bp; ETC1-II: −794 to −789 bp; ETC1-III: −232 to −227 bp; ETC3-I: −900 to −895 bp; ETC3-II: −857 to −852 bp; TCL2-I: −1,985 to 1,980 bp; TCL2-II: −1,962 to 1,957 bp; TCL2-III: −892 to −877 bp. The blue arrows indicate the primers used for ChIP-PCR assay. C, ChIP-PCR assays using 5-d-old whole wild-type or 35S-Myc-mTCP4 seedlings grown under normal conditions. The promoter regions containing TCP4-binding motif were amplified with the primer pairs named as in Figure 5B (blue arrows). The relative enrichment of the wild-type group was set to 1.0. The TA3 transposon locus was used as a negative control. The data are the means (±se) of three biological replicates. The asterisks indicate the Student’s t test significance. **P < 0.01; ***P < 0.001. D–G, The relative expression levels of R3 MYB genes (D: ETC1; E: ETC3; F: CPC; G: TCL2) in 3- to 7-d-old cotyledons of wild-type and tcp2/3/4/5/10/13/17 plants. The expression level of each gene in 3-d-old wild-type cotyledons was set as 1.0. ACTIN8 was used as the reference gene. The data are the means (±se) of three biological replicates. The asterisks indicate the Student’s t test significance. **P < 0.01; ***P < 0.001.

Figure 6

The expression pattern of ETC1 or ETC3 overlaps with that of TCP4, and overexpression of ETC1 or ETC3 rescues the ectopic trichome phenotype of tcp2/3/4/5/10/13/17. A–F, The expression patterns of ETC1 in 2- to 7-d-old wild-type cotyledons with GUS histochemical staining. The coding region of ETC1 was driven by its native promoter and fused with GUS gene. Bars = 200 μm. G–L, The expression patterns of ETC3 in 2- to 7-d-old wild-type cotyledons with GUS histochemical staining. The coding region of ETC3 was driven by its native promoter and fused with GUS gene. Bars = 200 μm. M–O, Overexpression of ETC1 or ETC3 rescued the ectopic trichomes on the cotyledons of the tcp2/3/4/510/13/17 mutant. White arrows indicate the trichomes on tcp2/3/4/510/13/17 cotyledons. Bars = 1 mm.

Figure 7

The working model of TCP transcription factors controlling trichome formation on cotyledons. A, The schematic representation of the working model of TCPs in repression of trichome formation in wild-type cotyledons. TCPs directly increase the expression levels of R3 MYB genes. R3 MYB proteins and TCPs per se inhibit the transactivation activity of the MBW complex by directly interacting with the components of MBW to suppress the expression of the downstream gene GL2, causing glabrous cotyledons after seed germination. B, The schematic representation of the working model of trichome formation on cotyledons of the tcp2/3/4/510/13/17 mutant. Loss of functions of TCPs result in the inactivation of CPC, ETC1, ETC3, and TCL2. The lack of TCPs and these R3 MYB proteins cause the expressed GL3, GL1, and TTG1 to form an active MBW complex, promoting trichome formation.