TOPLESS Regulates Apical Embryonic Fate in Arabidopsis

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Science  09 Jun 2006:
Vol. 312, Issue 5779, pp. 1520-1523
DOI: 10.1126/science.1123841


The embryos of seed plants develop with an apical shoot pole and a basal root pole. In Arabidopsis, the topless-1 (tpl-1) mutation transforms the shoot pole into a second root pole. Here, we show that TPL resembles known transcriptional corepressors and that tpl-1 acts as a dominant negative mutation for multiple TPL-related proteins. Mutations in the putative coactivator HISTONE ACETYLTRANSFERASE GNAT SUPERFAMILY1 suppress the tpl-1 phenotype. Mutations in HISTONE DEACETYLASE19, a putative corepressor, increase the penetrance of tpl-1 and display similar apical defects. These data point to a transcriptional repression mechanism that prevents root formation in the shoot pole during Arabidopsis embryogenesis.

The apical/basal axis of Arabidopsis embryos is established during the first cell division of the zygote, and auxin accumulation and response have been shown to be important for early steps in axis establishment (15). As the embryo matures, specific cell types become apparent, and a clear shoot/root axis is visible at the transition stage of development (6, 7). Although several mutants have been isolated that affect the formation of specific patterning elements of the shoot at the transition stage of embryogenesis, only topless-1 (tpl-1) so far switches the identity of the shoot into that of a root (811). It is therefore likely that TPL is acting at a different level of control than those factors that have previously been isolated.

tpl-1 mutants are temperature sensitive and at the restrictive temperature (29°C) transform the embryonic shoot pole into a second root pole that gives rise to a double-root seedling (11) (Fig. 1, A and B). At lower temperatures, tpl-1embryos fail to form a shoot apical meristem and show varying degrees of cotyledon fusion (Fig. 1, C to E). We view these phenotypes as a result of partial apical-to-basal transformation during embryogenesis (11) (fig. S1). Previous work has shown that transition-stage tpl-1 embryos lack or have reduced expression of genes associated with the apical half of the embryo, whereas the expression patterns of genes associated with the basal half of the embryo are expanded into the apical half and are ultimately duplicated. Pre–transition stage tpl-1 embryos are morphologically indistinguishable from those of the wild type.

Fig. 1.

Effects of topless-1 on embryonic polarity. (A) Wild-type 5-day-old seedling. (B) A tpl-1 double-root seedling. (C) A tpl-1 pin seedling lacking cotyledons. (D) A tpl-1 tube seedling. (E) A tpl-1 monocot seedling with two fused cotyledons. (F) WUS mRNA accumulation in a tpl-1 globular-stage embryo grown at 29°C. (G) WUS mRNA does not accumulate in a tpl-1 heart-stage embryo. (H) Wildtype heart-stage embryo accumulating WUS mRNA in a small group of cells in the developing meristem. Scale bars: 1 mm (A to E), 25 μm (F to H).

To examine the molecular organization of the apical half of tpl-1 pre–transition stage embryos, we performed in situ hybridizations with the transcription factor WUSCHEL (WUS) (10). WUS is initially expressed in a small group of cells in the apical half of 16-cell-stage embryos. WUS mRNA accumulated normally in tpl-1 globular-stage embryos, but was absent in transition-stage embryos at 29°C (Fig. 1, F to H). This indicates that early tpl-1 embryos have established an apical axis with the correct organization, but this fate is lost or masked at the transition stage.

tpl-1 was mapped to bacterial artificial chromosome F7H2 on chromosome 1 using polymerase chain reaction–based markers (11). We found two base-pair substitutions in At1g15750 (12) that cosegregated with the tpl-1 phenotype and result in a change of a lysine (K) to a methionine (M) at amino acid 92 and an asparagine (N) to a histidine (H) at amino acid 176 of the predicted protein (13). Concurrently, we conducted a high-temperature ethylmethane sulfonate suppressor screen in the tpl-1 background and found five semidominant suppressors that mapped to the original TPL locus. We sequenced At1g15750 from these lines and found that each harbored a second site mutation that is predicted to reduce or abolish gene function (Fig. 2A). That second site mutations in the tpl-1 mutant gene suppress the tpl-1 phenotype indicates that tpl-1 is a gain-of-function allele. The semidominant nature of these loss-of-function alleles also implies a dosage requirement for the tpl-1 protein.

Fig. 2.

Molecular characterization of the TPL gene. (A) Diagram of the predicted structure of the TPL protein. TPL is predicted to have a LisH (blue circle) and CLISH (green hexagon) domain at the N terminus, a 100–amino acid proline-rich domain (yellow box), and 11 WD40 repeats (red boxes). The tpl-1 phenotype is caused by a substitution of an asparagine at amino acid 176 with a histidine. tpl-2 and tpl-3 are splice acceptor site mutations, and tpl-4 is a splice donor mutation. tpl-5 is caused by a substitution of a serine at amino acid 578 with a phenylalanine in the sixth WD40 repeat, and tpl-6 is caused by a change of a glutamine at amino acid 991 to a stop codon (CAA to TAA). tpl-8 is a T-DNA insertion allele (SALK_036566). Numbers represent the affected amino acid positions. AA, amino acids. (B and C) TPL mRNA accumulation in (B) a globular-stage and (C) torpedo-stage wild-type embryo. (D) A translational fusion of TPL to GFP localizes to the nuclei of all cells in a four-cell-stage embryo. (E)A tpl-2 mutant shows no phenotype after developing at 29°C. (F) A tpl-2; tpr1-1; tpr3-1;tpr4-1 mutant carrying a TPR2 RNAi construct displaying a pin phenotype. Scale bars: 25 μm(BtoD), 1 mm (E and F).

TPL is predicted to encode an 1131–amino acid protein containing 11 WD40 repeats at the C terminus (Fig. 2A). At the N terminus, TPL has predicted lissencephaly type 1–like homology (LisH) and C-terminal to LisH (CTLH) domains that are thought to be important either for self-dimerization or for other protein-protein interactions (14). TPL also contains a 100–amino acid region rich in prolines (24 out of 100 amino acids). A similar domain organization is found in the TUP1/GROUCHO and LEUNIG family of transcriptional corepressors, although there is little sequence identity between TPL and these proteins (15, 16). Four other predicted proteins in Arabidopsis share extensive amino acid similarity with TPL and have been named TOPLESS-RELATED (TPR) (fig. S2).

In situ hybridization experiments revealed that TPL mRNA accumulates in all cells of the embryo as well as in extra-embryonic tissues (Fig. 2, B and C). TPL mRNA accumulates to higher levels in the embryo proper during early embryogenesis and in the developing vasculature in later stages. A TPL-GREEN FLUORESCENT PROTEIN (GFP) translational fusion under the control of 4.1 kb of upstream genomic sequences rescued the tpl-1 phenotype when homozygous and localized to the nuclei of all cells in transgenic plants (Fig. 2D). This again indicates a dosage dependence for the tpl-1 protein and suggests that the wild-type version of the protein can outcompete the mutant form.

To determine if both of the amino acid changes found in the original tpl-1 allele were necessary for the tpl-1 phenotype, we transformed a tpl transfer DNA (T-DNA) insertion line (tpl-8) with TPL-GFP fusion proteins containing either both mutations (tpl-1), only the K92M mutation, or only the N176H mutation (17). The tpl-1 phenotype was observed in plants carrying either the tpl-1 transgene or the N176H transgene (16 and 15 lines, respectively). However, we did not observe any tpl phenotypes in 29 independent lines transformed with the K92M construct despite nuclear GFP accumulation comparable to that of lines with a tpl-1 phenotype. Therefore, the N176H mutation is necessary and sufficient to cause the tpl-1 phenotype.

tpl loss-of-function alleles display no obvious phenotype when grown at the restrictive temperature (Fig. 2E). We therefore hypothesized that TPL may act redundantly with the other TPR proteins. We generated tpl-2; tpr1; tpr3; tpr4 quadruple mutant lines and transformed them with a TPR2 RNA interference (RNAi) transgene. We obtained five stable transgenic lines that displayed the original tpl-1 phenotypes (Fig. 2F). This indicates that tpl-1 acts as a type of dominant negative allele for multiple TPR family members.

In the high-temperature suppressor screen, we also isolated two alleles of a recessive extragenic suppressor of tpl-1 designated big top (bgt). At 24°C, the progeny of plants homozygous for tpl-1 and heterozygous for bgt-1 segregated 24.1% wild-type seedlings (Fig. 3B) (n = 513). This same combination with bgt-2 yielded 19.3% wild-type seedlings (n = 1746). We therefore characterized bgt-1 in more detail. Morphologically, tpl-1; bgt-1 embryos form cotyledons at the transition stage of embryogenesis, although they appear slightly stunted at later stages as compared to wild-type embryos (Fig. 3, C and D). To examine the apical pattern of tpl-1; bgt-1 embryos, we examined the expression of WUS in these double mutants at 29°C. At all stages tested, tpl-1;bgt-1 embryos maintained the expression of WUS in the appropriate number of cells, indicating that the top half of these embryos had not lost their apical identity (Fig. 3, E to G).

Fig. 3.

Characterization of hag1 alleles and genetic interactions with tpl-1. (A) Diagram of the predicted structure of HAG1 that contains a conserved histone acetyltransferase domain (red box) and a bromo domain (yellow box). hag1-3 contains a stop codon at amino acid 478 (TGG to TGA); hag1-4 contains a splice donor mutation at amino acid 389; hag1-5 is a T-DNA insertion in the 10th intron (SALK_048427); and hag1-6 is a T-DNA insertion in the first intron (SALK_150784). (B) A tpl-1;hag1-3 doublemutant seedling grown at 24°C. (C and D) Cleared torpedo-stage embryos of (C) tpl-1;hag1-3 and (D) wildtype seedling grown at 29°C. (E to G) WUS mRNA accumulation in (E) tpl-1, (F) tpl-1;hag1-3, and (G) wild-type 29°C grown torpedo-stage embryos. (H) A HAG1-GFP fusion protein localizes to the nuclei of all cells of a 16-cell-stage embryo. Scale bars: 1 mm (B), 25 μm (C to H).

We mapped the bgt-1 mutation and found that it was tightly linked to marker TSA1 on chromosome 2 (0 recombinants out of 606 chromosomes). This genomic region contains the Arabidopsis homolog of the histone acetyltransferase GCN5 (HAG1) (also known as atGCN5) (18, 19). In other eukaryotes, GCN5 is recruited to specific promoters by DNA binding transcription factors and is thought to promote transcription by acetylating the N-terminal tail of histone H3 (20). Sequencing revealed that both bgt-1 and bgt-2 carried lesions in HAG1 (Fig. 3A). We therefore renamed these alleles hag1-3 and hag1-4. T-DNA insertions in the tenth intron (hag1-5) and in the first intron (hag1-6) also suppressed tpl-1 (Fig. 3A). All four hag alleles have no obvious embryonic phenotypes, although postembryonically they display pleiotropic phenotypes similar to that of a previously described allele (18). A translational fusion of a 4.3-kb HAG1 genomic clone to GFP rescued the hag1-3 mutant, and the protein was found in the nuclei of all cells examined (Fig. 3H). The observation that a mutation in a coactivator suppresses the tpl-1 phenotype is consistent with TPL acting as a corepressor.

In eukaryotes, transcription from many promoters can be repressed through the activity of histone deacetylases. The RPD3 family of histone deacetylases can act as transcriptional corepressors, and in Drosophila, Groucho and an RPD3-like protein work together to specify anterior/posterior polarity (21). The Arabidopsis genome contains four class 1 RPD3-like proteins [Histone Deacetylase (HDA) 6, 7, 9, and 19] (22). In a screen for mutants that affect floral organ identity, a T-DNA allele of HDA19 (hda19-1) (also known as atHD1 and RPD3a) was isolated that displays floral phenotypes similar to those of tpl-1 (2325). A second T-DNA allele (hda19-2) was isolated from the Wisconsin Arabidopsis Knockout facility and found to show similar phenotypes (Fig. 4A). We therefore examined the role and expression of HDA19 more closely during embryogenesis.

Fig. 4.

Characterization and genetic interactions of HDA19. (A) Predicted structure of HDA19. hda19-1 contains a T-DNA insertion that disrupts amino acid 312 in the histone deacetylase domain (red box). hda19-2 contains a T-DNA insertion 5 base pairs upstream of the start codon. (B) mRNA accumulation of HDA19 in all cells of an early heart-stage embryo. (C) A HDA19-GFP fusion protein localizes to the nuclei of all cells of a 16-cell-stage embryo. (D) Seedling phenotype of hda19-1 when grown at 24°C. (E) A hda19-2 seedling displaying a pin phenotype when grown at 29°C. (F) A hda19-1 heart-stage embryo grown at 29°C showing both shoot and root defects. (G) A tpl-1;hag1-3;hda19-1 triple-mutant seedling grown at 24°C. Scale bars: 25 μm(B,C, and F), 1 mm (D, E, and G).

HDA19, like TPL and HAG1, is broadly expressed throughout embryogenesis, and a GFP fusion protein localizes to the nuclei of all embryonic cells (Fig. 4, B and C). Phenotypically, both hda19-1and hda19-2 seedlings when grown at 24°C have narrow cotyledons as compared to those of the wild type (Fig. 4D). However, when mutants homozygous for either allele were grown at 29°C, mutant seedlings displayed several tpl-1–like phenotypes, including monocots, tubes, and pins, indicating that these hda19 alleles are temperature sensitive (Fig. 4E). These phenotypes were seen in 32% of hda19-1 seedlings (n = 397) and 28% of hda19-2 seedlings (n = 330). A morphological analysis of hda19-1 embryos at 29°C showed that both the root and the shoot can be disorganized (Fig. 4F), indicating that HDA19 may play a broader role in embryogenesis than TPL.

We then examined the progeny of hda19-1/; tpl-1+/ plants grown at 24°C, a temperature at which tpl-1 segregates as a recessive (11). We found that 45% of the resulting seedlings showed cotyledon fusion defects (n = 804) instead of the expected 25%, indicating that HDA19 may act on some of the same target genes as TPL during embryogenesis. In agreement with this hypothesis, we identified tpl-1; hda19-1; hag1-3 triple-mutant seedlings from plants grown at 24°C, as well as 29°C, and found that they displayed two narrow cotyledons like the hda19-1 single mutant (Fig. 4G). Therefore, hag1-3 mutants can suppress tpl-1 mutant phenotypes even in the absence of functional HDA19.

Recent work on embryonic polarity in Arabidopsis has focused on auxin transport and the first embryonic cell divisions in establishing the apical/basal axis (4, 26). Our studies have uncovered a set of proteins involved in a new step in axis formation, during the transition stage of embryogenesis, when shoot fate becomes fixed and distinct from root fate. We propose that at the transition stage of embryogenesis, TPL and other TPR proteins are necessary to repress the expression of root-promoting genes in the top half of the embryo to allow proper differentiation of the shoot pole. A histone deacetylase, HDA19, works in conjunction with TPL during this process, although it appears to have TPL-independent roles as well (27). HAG1 is necessary for the complete transformation of the apical half into a root, likely by activating the transcription of derepressed root-specific genes in the apical half of the embryo. However, HAG1 is dispensable for the formation of the basal “true” root. Conceptually, these two steps of polarity determination are similar to what has been reported in the brown alga Fucus, where axis formation and fixation are temporally distinct (28). In Arabidopsis, we propose that the axis formation step occurs during the first cell divisions of the embryo and likely relies on polar auxin distribution (4). Only later, at the transition stage of embryogenesis, does the axis become fixed, at which time the plant requires a chromatin-mediated transcriptional repression system for axis stabilization.

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Materials and Methods

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