The TRANSPARENT TESTA16 locus encodes the ARABIDOPSIS BSISTER MADS domain protein and is required for proper development and pigmentation of the seed coat

Abstract

Screening for seed pigmentation phenotypes in Arabidopsis led to the isolation of three allelic yellow-seeded mutants, which defined the novel TRANSPARENT TESTA16 (TT16) locus. Cloning of TT16 was performed by T-DNA tagging and confirmed by genetic complementation and sequencing of two mutant alleles. TT16 encodes the ARABIDOPSIS BSISTER (ABS) MADS domain protein. ABS belongs to the recently identified "B-sister" (B(S)) clade, which contains genes of unknown function that are expressed mainly in female organs. Phylogenetic analyses using a maximum parsimony approach confirmed that TT16/ABS and related proteins form a monophyletic group. TT16/ABS was expressed mainly in the ovule, as are the other members of the B(S) clade. TT16/ABS is necessary for BANYULS expression and proanthocyanidin accumulation in the endothelium of the seed coat, with the exception of the chalazal-micropylar area. In addition, mutant phenotype and ectopic expression analyses suggested that TT16/ABS also is involved in the specification of endothelial cells. Nevertheless, TT16/ABS apparently is not required for proper ovule function. We report the functional characterization of a member of the B(S) MADS box gene subfamily, demonstrating its involvement in endothelial cell specification as well as in the increasingly complex genetic control of flavonoid biosynthesis in the Arabidopsis seed coat.

Figure 1.

Main Routes for Flavonoid Biosynthesis in Arabidopsis. The pathway is initiated by the condensation of 4-coumaroyl-CoA (derived from phenylpropanoid metabolism) with malonyl-CoA. The three major end products of flavonoid metabolism in Arabidopsis are presented with indications of their respective color and localization. Enzymes are designated in capital letters. Mutants corresponding to genes of known function are indicated in lowercase italic letters. Dotted lines indicate different unidentified steps. ban, banyuls; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; LAR, leucoanthocyanidin reductase; tt, transparent testa.

Figure 2.

Seed Pigmentation Phenotypes. (A) to (C) Mature seeds from a wild-type plant (A), a tt16-1 homozygous plant (B), and the T2 progeny of a tt16-1 homozygote transformed with the TT16/ABS gene under the control of the double-enhanced 35S promoter (35S²::TT16/ABS) (C). White arrows in (A) and (B) point to the chalaza-micropyle area in mature seeds. tt16-1 seeds are larger than wild-type seeds and occasionally display premature germination (black arrowhead in [B]). All genotypes are from the Wassilewskija-2 ecotype. Bar in (A) = 200 μm for (A) to (C). (D) to (G) PA deposition pattern in immature seeds was visualized with vanillin staining. Genotypes shown in (D), (E), and (F) and (G) correspond to the genotypes shown in (A), (B), and (C), respectively. Seeds were approximately at the globular stage of embryo development. The endothelium layer of tt16-1 seed does not exhibit PA accumulation, except in the chalazal-micropylar area (E). Ectopic vanillin staining is shown in seeds carrying the 35S²::TT16/ABS construct (black arrowheads in [F] and [G]). Bar in (D) = 30 μm for (D) to (F) and ∼15 μm for (G). (H) and (I) Histochemical analysis of GUS reporter gene expression driven by the BAN promoter in seeds from the wild type (H) and the tt16-1 mutant (I). GUS expression patterns in (H) and (I) fully mimic the PA deposition profiles shown in (D) and (E), respectively. Seeds were at the early heart stage of embryo development. Photographs were made with phase-contrast optics. Bar in (H) = 35 μm for (H) and (I). cb, chalazal bulb; eb, endothelium body; emb, embryo; en, endothelium; mi, micropyle; WT, wild type.

Figure 3.

Characterization of the TT16/ABS Gene. (A) Structure of the TT16/ABS gene and positions of tt16 mutations. Exons are represented by closed rectangles with their sizes indicated in brackets, and introns are represented by lines. The positions and the nature of mutations in tt16 alleles are shown at top. T-DNA insertion in tt16-1 causes a deletion of plant genomic DNA, as indicated by the crosshatched box. The exon-intron structure of the TT16/ABS gene in tt16-3 is detailed below the scheme. Insertion of a fragment from T-DNA creates two novel exons (3′ and 3′′, respectively) and introduces a premature termination codon (star). Primers used for molecular analyses are indicated. Numbering of the nucleotide positions is given in base pairs from the translational start codon. The conserved MADS domain found in TT16/ABS is shown by the solid bar. LB, left border; RB, right border. (B) Donor and acceptor splicing sites of intron 3 in the wild-type TT16/ABS gene. Alternative splicing occurs at the 3′ end of intron 3, leading to two cDNA molecules that differ by 15 bp (highlighted in boldface letters in the longer cDNA sequence). The amino acid residues deduced from the translation of each cDNA sequence are given below the corresponding codons.

Figure 4.

Comparison of TT16/ABS with Other Related MADS Domain Proteins from the B_S Clade. (A) Alignment of deduced amino acid sequences for Arabidopsis TT16/ABS and related proteins from petunia (FBP24), A. majus (DEFH21), maize (ZMM17), and G. gnemon (GGM13). The MADS, I, K, and PI domains are indicated by arrows. Identical amino acid residues are boxed, and similar residues are shaded in gray. Dashes indicate gaps in the sequences to allow maximal alignment. The intron positions in TT16/ABS are shown above the alignment (I-1 to I-5). Putative base-contacting residues in the MADS domain of TT16/ABS are indicated by closed squares. The two putative amphipathic α-helices in the K domain are highlighted by dotted lines. (B) Phylogenetic relationships between TT16/ABS and related MADS domain proteins. Shown is a simplified cladogram illustrating the consensus most-parsimonious pattern of relationships obtained using weighted MP analysis (see Methods). The monophyletic origin of the B_S clade is presented with that of FBP24 (petunia), DEFH21 (A. majus), TT16/ABS (Arabidopsis), ZMM17 (maize), and GGM13 (G. gnemon). Bootstrap percentages are indicated before nodes, and nodes with a bootstrap score of <50% were discarded.

Figure 5.

RT-PCR Showing TT16/ABS mRNA Accumulation in Organs of Arabidopsis Plants. TT16/ABS transcripts were detected after 35 cycles of PCR amplification on reverse transcription products using primers TT16-6 and TT16-Stop (see Figure 3A). PCR products were visualized on a 2.5% (w/v) agarose gel stained with ethidium bromide. The two bands correspond to the two cDNA forms. Transcript levels of the Arabidopsis elongation factor EF1αA4 were used as a control (migrated on a 1% [w/v] agarose gel). Abbreviations for the organs examined are as follows: Bd, floral buds; Fl, full-blown flowers; Sil, immature siliques at different ages of embryo development; Se, seeds; SilV, silique valves; Sd, 4-day-old seedlings; St, stems; Le, rosette leaves; Rt, roots. For silique samples, Sil1 comprises siliques from 1 and 2 days after pollination (dap), Sil2 comprises siliques from 3 to 5 dap, and Sil3 comprises siliques from 6 to 10 dap. Seeds in lane 6 were harvested from the globe to late-torpedo stages of embryo development and removed from the silique valves.

Figure 6.

Structure of the Seed Coat. (A) to (E) Sections (2 μm thick) through immature seeds after staining with toluidine blue. Ovules in (A) and (B) were taken at the one-celled stage of embryo development (before pigment deposition). Seeds in (C) to (E) were harvested at the globular embryo stage. The five layers of the seed coat are indicated in (C), according to Beeckman et al. (2000). Arrowheads in (B) and (D) show flattened and irregular cells in tt16-1 endothelium. (E) represents an enlarged view of the micropylar zone in tt16-1 seed, and the remaining pigmentation is indicated by arrows. (C) is reprinted from Nesi et al. (2001). Bars = 5 μm for (A) and (B) and 20 μm for (C) and (D). (F) to (O) Cleared seeds viewed with Nomarski optics. Comparison of the development of the wild-type and tt16-1 seed coat, with an emphasis on the development of the endothelium. (F) and (G) Early development of the two ovule integuments. (H) and (I) Prefertilization stages with mature embryo sacs. (J) to (L) Globular stage of embryo development. (M) to (O) Seeds at the heart-shaped embryo stage. The endothelium is indicated by white arrows ([H] to [O]). (L) and (O) show seeds carrying the 35S²::TT16/ABS construct. Bars = 5 μm for (F) and (G), 20 μm for (H) and (I), 25 μm for (J) to (L), and 30 μm for (M) to (O). cb, chalazal bulb; en, endothelium; ii, inner integument; mi, micropyle; oi, outer integument; WT, wild type.

Figure 7.

Effects of TT16/ABS Ectopic Expression on Plant Phenotype. Phenotypes of plants carrying the 35S²::TT16/ABS construct ([B], [C], [E], and [F]) compared with nontransformed wild-type plants ([A], [D], and [F]). Approximately 50% of the 35S²::TT16/ABS transgenic lines showed curled leaves ([B] and [C]), reductions in flower size (E), and shrunken siliques (F). Plants in (A), (B), and (C) are shown at ∼20, 30, and 60 days after sowing, respectively. WT, wild type. Bars = 0.5 cm.