STENOFOLIA recruits TOPLESS to repress ASYMMETRIC LEAVES2 at the leaf margin and promote leaf blade outgrowth in Medicago truncatula

Abstract

The Medicago truncatula WUSCHEL-related homeobox (WOX) gene, STENOFOLIA (STF), plays a key role in leaf blade outgrowth by promoting cell proliferation at the adaxial-abaxial junction. STF functions primarily as a transcriptional repressor, but the underlying molecular mechanism is unknown. Here, we report the identification of a protein interaction partner and a direct target, shedding light on the mechanism of STF function. Two highly conserved motifs in the C-terminal domain of STF, the WUSCHEL (WUS) box and the STF box, cooperatively recruit TOPLESS (Mt-TPL) family corepressors, and this recruitment is required for STF function, as deletion of these two domains (STFdel) impaired blade outgrowth whereas fusing Mt-TPL to STFdel restored function. The homeodomain motif is required for direct repression of ASYMMETRIC LEAVES2 (Mt-AS2), silencing of which partially rescues the stf mutant phenotype. STF and LAMINALESS1 (LAM1) are functional orthologs. A single amino acid (Asn to Ile) substitution in the homeodomain abolished the repression of Mt-AS2 and STF's ability to complement the lam1 mutant of Nicotiana sylvestris. Our data together support a model in which STF recruits corepressors to transcriptionally repress its targets during leaf blade morphogenesis. We propose that recruitment of TPL/TPL-related proteins may be a common mechanism in the repressive function of modern/WUS clade WOX genes.

Figure 1.

The HD and CTD of STF Are Essential for Blade Outgrowth Function. (A) Schematic representation of domain arrangements in STF protein from the N terminus to the C terminus. Domains are as follows: NTD, amino acids 1 to 90; HD, amino acids 91 to 163; MD, amino acids 164 to 300; CTD, amino acids 301 to 358. The CTD contains two conserved motifs: the WUS box (yellow; amino acids 309 to 318, QTLQLFPIRN) and the STF box (blue; amino acids 349 to 358, QFIEFLPLKN). (B) Constructs used in the deletion assay for complementation of the N. sylvestris lam1 mutant. All constructs were driven by the M. truncatula STF promoter. (C) to (H) Complementation of N. sylvestris lam1 by deletion mutant constructs of the M. truncatula STF gene. The lam1 mutants were transformed with STF:STF (C), STF:HD-MD-CTD (D), STF:NTD-HD-CTD (E), STF:HD-CTD (F), STF:MD-CTD (G), and STF:STFdel (H). Bars = 5 cm.

Figure 2.

Both the WUS Box and STF Box in the CTD Are Required for STF Function. (A) Schematic representation of reporter and effector constructs used in the luciferase assay. Red and blue lines represent the m1 and m2 mutations, respectively, shown in (B). (B) Leu (L) to Ala (A) substitution mutations in the CTD, yielding m1 in the WUS box (WB) and m2 in the STF box (SB). (C) Relative luciferase activities using STF, STFm1, STFm2, STFm1m2, or STFdel as effector compared with the GAL4-DB control. Error bars indicate sd (n = 3). **P < 0.01 (t test). (D) Constructs used for complementation of the lam1 mutant. Red and blue lines represent m1 and m2 mutations, respectively. (E) to (H) lam1 mutant plants and leaves complemented with STF:SRDX-STFdel (E), STF:STFm1 (F), STF:STFm2 (G), and STF:STFm1m2 (H). Bars = 5 cm.

Figure 3.

The STF Box Is Evolutionarily Conserved in Dicots and Plays a Stronger Role Than the WUS Box. (A) Constructs used for complementation of the lam1 mutants. Either WUS box (WB) or STF box (SB) was fused in-frame to the CTD-truncated mutant of STF (STFdel). (B) Amino acid sequence alignment of the STF box in STF and STF-like (STL) proteins from dicot species. A consensus FxEFLP motif is shown at the bottom. Ms, Medicago sativa; Gm, Glycine max; Lj, Lotus japonicus; Csi, Citrus sinensis; Vv, Vitis vinifera; Rc, Ricinus communis; Cp, Carica papaya; Nb, N. benthamiana; Ns, Nicotiana sylvestris; Pxh, Petunia × hybrida; Eg, Eucalyptus grandis; St, Solanum tuberosum; Sl, Solanum lycopersicum; Cm, Cucumis melo; Gr, Gossypium raimondii; Aq, Aquilegia coerulea; Mg, Mimulus guttatus; At, Arabidopsis; Al, Arabidopsis lyrata; Amt, Amborella trichopoda. (C) Leu (L) to Ala (A) mutations in STF box and WUS box. (D) to (I) Phenotypes of transgenic lam1 plants complemented with STF:STFdel-WUS-box (D), STF:STFdel-STF-box (E), STF:STFm1-m2L1A (F), STF:STFm1-m2L2A (G), STF:STFm1L1A-m2 (H), and STF:STFm1L2A-m2 (I). Bars = 5 cm.

Figure 4.

STF Physically Interacts with Mt-TPL through Its WUS Box and STF Box and This Interaction Is Required for Leaf Blade Outgrowth. (A) Interaction between STF and Mt-TPL in the Y2H assay. Interaction was examined by the presence or absence of yeast growth on a quadruple dropout medium (QDO)/X-Gal plate. DDO, double dropout medium. (B) Interaction between STF and Mt-TPL in living cells using a split YFP BiFC assay. Bar = 50 μm. (C) Interaction between STF and Mt-TPL in pull-down assay. The TPL-GFP fusion protein, transiently expressed in tobacco leaves, was pulled down by amylose resin–immobilized MBP protein or MBP-STF fusion protein and detected by anti-GFP antibody (α-GFP). Immobilized MBP protein or MBP-STF fusion protein was stained by Coomassie blue and shown as bait. (D) Mapping of the STF domains required for interaction with Mt-TPL. Different STF mutants were fused to the GAL4-DBD domain, while TPL was fused to the GAL4-AD domain. EV, empty vector. Interaction was examined by yeast growth on triple dropout medium (TDO). Data are representative of three repeats. (E) BiFC showing the absence of interaction between STFm1m2 and TPL in living cells. Bar = 50 μm. (F) Mapping of the Mt-TPL domains required for interaction with STF. (G) Y2H interaction of the TPL mutant constructs in (F) with STF. (H) Constructs used for complementation of lam1 mutants. (I) to (K) Phenotypes of lam1 mutant plants complemented with STF:STFdel (I), STF:STFdel-TPL (J), and STF:TPL (K). Bars = 5 cm.

Figure 5.

STF Represses AS2 Expression at the Leaf Margin in M. truncatula. (A) Structure of the STF HD based on template 2da3A. A point mutation (m3, Asn to Ile) in position 147 is shown. (B) N. sylvestris lam1 mutant complemented with mutated Mt-STFm3. The N147I mutation abolished the STF activity in complementing the lam1 mutant. (C) Relative expression level of AS2 in the M. truncatula wild type, stf mutant, and STF overexpression lines (OX). The expression level of AS2 in the wild type was set to 1.0. Error bars indicate sd (n = 3). **P < 0.01 (t test, compared with the wild type). (D) to (F) RNA in situ hybridization showing Mt-AS2 expression viewed in longitudinal sections of 4-week-old shoot apices in the wild type (D) and the stf mutant (E). A negative control is shown in (F) using sense probe. Arrows in (D) and (E) indicate the absence or presence of AS2 signal at the leaf margin in wild-type and stf mutant leaf primordia, respectively. Bars = 50 μm. (G) A cartoon depicting the expression pattern of AS2 (red) in wild-type and stf mutant leaf primordia observed in (D) and (E). Note that AS2 expression is excluded from the leaf margin in the wild type, while expression is extended beyond the margin into the abaxial domain in the stf mutant.

Figure 6.

STF Directly Binds to the Mt-AS2 Upstream Promoter Region. (A) Schematic representation of the proAS2:AS2 construct, which complemented the Arabidopsis as2-1 asymmetric leaf phenotype. The promoter regions tested by ChIP assays are indicated as P1, P2, P3, and P4. The Mt-AS2 coding region (from the translation start site ATG) is shown by the thick line, and the CDS region tested by the ChIP assay is designated as 5. (B) ChIP assays. The proAS2:AS2/as2-1 leaf protoplasts were transfected by pro35S:STF-YFP. Chromatin complexes were cross-linked by formaldehyde, fractioned by sonication, immunoprecipitated by anti-GFP antibody or anti-HA antibody, and collected by protein A agarose beads. The purified DNAs were used as templates for PCR. PCR products for P1, P2, P3, and P4 were enriched in anti-GFP samples compared with the anti-HA samples. For the Mt-AS2 coding region (5) and the Arabidopsis ACTIN gene (Ac), no PCR products were detected. Similar results were obtained from three biological replicates. (C) EMSA showing MBP-STF (S) bound to the biotin-labeled P2 promoter fragment but not to the MBP (M) control. Fifty times (50×) unlabeled P2 DNA completely competed out the binding (right lane). (D) EMSA showing MBP-STF (S) bound to the biotin-labeled P3 promoter fragment but not to the MBP (M) control. Fifty times (50×) unlabeled P3 DNA competed out this binding to a large extent. (E) Compared with the MBP-STF fusion protein (S), which bound to the P2 and P3 promoter fragments, the MBP-STFm3 fusion protein (Sm3) lost the ability to bind DNA, confirming that the Asn-147 site of the HD is critical for STF interaction with the AS2 promoter. (F) EMSA showing MBP-STF (S) bound to the biotin-labeled P4 promoter fragment but not to the MBP (M) control or the m3 mutant (Sm3).

Figure 7.

The STF-TPL Complex Represses Mt-AS2 Promoter Activity. (A) Schematic representation of reporter and effector constructs used in the luciferase assay. The 3-kb Mt-AS2 promoter region was fused to a mini 35S promoter to drive the expression of the luciferase reporter gene. Elements of the scheme are not drawn to scale. (B) Relative luciferase activities in Arabidopsis protoplasts using STF, STFm3, or STFdel as effector compared with the GUS control. Error bars indicate sd (n = 3). **P < 0.01 (t test). (C) Relative luciferase activities using Mt-STF as effector compared with the GUS control in Arabidopsis wild-type (Ler) or tpl-1 mutant protoplasts. Error bars indicate sd (n = 3). **P < 0.01 (t test). (D) Complementation of M. truncatula stf mutant leaf blade phenotypes by STF:STFdel (1), STF:STF (2), and STF:STFdel-TPL (3) constructs. (E) Relative expression of AS2 determined by quantitative RT-PCR in the stf mutant background complemented with STFdel (1), STF:STF (2), and STF:STFdel-TPL (3) compared with the wild type. The expression level of AS2 in the wild type was set to 1.0. Error bars indicate sd (n = 3). **P < 0.01 (t test). [See online article for color version of this figure.]

Figure 8.

Silencing of AS2 by RNAi in M. truncatula and Overexpression of Mt-AS2 in N. sylvestris. (A) Silencing of AS2 in wild-type M. truncatula leaves caused epinastic (top panel) and bigger (bottom panel) blades. Leaves of two representative RNAi lines are shown. (B) Silencing of AS2 in the M. truncatula stf mutant led to partial complementation of the leaf blade phenotype (right) compared with the mutant (middle) and the wild type (left). (C) Wild-type N. sylvestris control plant. (D) to (F) Phenotypes of N. sylvestris transgenic lines expressing the 35S:AS2 transgene showing severe upward curling and needle-like blades. Three representative transgenic lines are shown. (G) Untransformed N. sylvestris lam1 mutant (left) and lam1 mutant transformed with the 35S:AS2 transgene showing shorter and more erect leaves than lam1 (right). [See online article for color version of this figure.]