TRAUCO, a Trithorax-group gene homologue, is required for early embryogenesis in Arabidopsis thaliana

Abstract

Embryogenesis is a critical stage during the plant life cycle in which a unicellular zygote develops into a multicellular organism. Co-ordinated gene expression is thus necessary for proper embryo development. Polycomb and Trithorax group genes are members of evolutionarily conserved machinery that maintains the correct expression patterns of key developmental regulators by repressing and activating gene transcription. TRAUCO (TRO), a gene homologous to the Trithorax group of genes that can functionally complement a BRE2P yeast mutant, has been identified in Arabidopsis thaliana. It is demonstrated that TRO is a nuclear gene product expressed during embryogenesis, and loss of TRO function leads to impaired early embryo development. Embryos that arrested at the globular stage in the tro-1 mutant allele were fully rescued by a TRO expression clone, a demonstration that the tro-1 mutation is a true loss-of-function in TRO. Our data have established that TRO is the first trithorax-group gene homologue in plants that is required for early embryogenesis.

Fig. 1.

The Arabidopsis thaliana TRO is a Trithorax group homologous gene. (a) A schematic representation of the exon–intron structure of the genomic TRO gene with the location of the T-DNA insertion. (b) Phylogenetic tree analysis of TRO with TrxG genes from other organisms and with Arabidopsis SPRY-domain containing proteins. The tree was constructed by the Neighbor–Joining method with MEGA program 3.0. Branch numbers represent the percentage of bootstrap values in 1000 sampling replicates. The protein accession numbers are: TRO (NP175556), SEPR11 (ACE95183), ASH2 (AAC47328), ASH2-L (Q9UBL3), D. rerio ASH2-L (NP001103575), X. laevis ASH2-L (AAI55932), BRE2P (NP013115), At4G09200 (NP192659), At4G09310 (NP192669), and At4G09340 (NP192672). Sequences of the grape (CAO21886) and rice (Os11g0146500 and Os12g0143200) homologous genes were obtained from NCBI GeneBank. (c) Organization and comparison of TRO with homologous proteins. Locations of conserved domains with significant homologies are indicated by boxes: SPRY (light gray) and PHD (black). The percentages indicate the degree of amino acid similarity with respect to TRO for each conserved domain. Numbers below each protein indicate the initial and final amino acid of the corresponding domains.

Fig. 2.

Expression of TRO provides formamide tolerance to a bre2p yeast strain. Yeast cells were grown to an OD₆₀₀ nm value of 1.0, and then 5 μl of 10-fold serial dilutions (left to right in each panel) were spotted onto an solid media containing 0 (control) or 2.5% formamide according to Nagy et al. (2002). Growth was recorded after 10 d of culture. Yeast cells transformed with empty vector used as control (Bre2pΔ).

Fig. 3.

Gene expression of TRO in various plant tissues. Data used for the analysis were retrieved from GENEVESTIGATOR (Zimmermann et al., 2004). The values shown are means +SD.

Fig. 4.

Temporal and spatial patterns of TRO gene expression. (a–d) RNA in situ hybridization confirmed the expression of TRO during early embryogenesis. (a) Nomarski micrographs showing a strong signal in the embryo proper (EP) and suspensor (S) at the eight-cell stage. (b) Hybridization with a sense probe at the eight-cell stage. (c) Nomarski micrographs showing a strong signal at the heart stage. (d) Hybridization with a sense probe at the heart stage. (e–g) Histochemical assays of the expression pattern of GUS under the control of the TRO promoter in P_TRO–GUS transgenic Arabidopsis. (e) GUS staining of a 7-d-old seedling showing TRO expression in the cotyledons, shoot base and root. (f) GUS staining of a 14-d-old seedling showing TRO expression in cotyledons and hydathodes in a rosette leaf. (g) GUS staining in adult flowers showing TRO expression in pollen grains (inset), anthers, and sepals. Scale bar: a–d, 20 μm; e–f, 1 mm; g, 0.5 mm.

Fig. 5.

Subcellular localization of TRO. (a) Epifluorescence micrograph of an onion cell transiently expressing GFP (35S::GFP). (b) Nomarski micrographs showing the same cell in (a). (c) Epifluorescence micrograph of an onion cell transiently expressing GFP fused to TRO (35S::TRO-GFP). (d) Nomarski micrographs showing the same cell as (c). The white arrow highlights the nucleus. (Scale bar: 20 μm.

Fig. 6.

Mutant tro-1 embryos were arrested before the globular stage. (a) Wild-type silique showing a full seed set and a (b) heterozygous tro-1 silique with approximately 25% of the embryos aborted (black arrows). (c) Whole-mounted, cleared seeds from siliques of heterozygous tro-1 plants. The same silique contains a mutant embryo arrested at the early globular stage (left) compared with a normal embryo developed at the heart stage (right). (d–h) Nomarski images of wild-type embryos at the four-cell stage (d), eight-cell stage (e), early heart stage (f), heart stage (g), and torpedo (h). (i–m) Nomarski images of homozygous tro-1 embryos arrested within the same siliques as the wild-type embryos showed in (d–h). Mutant embryo with normal appearance at four-cell stage (i) and eight-cell stage (j). (k–m) Embryos with abnormal morphology compared with sibling embryos shown in (f–h), respectively. Scale bar: d–f and i–m, 20 μm; g, 25 μm; h, 40 μm.