The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques

Abstract

The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

Figure 1.

Outline of the Flavonoid Metabolism in Arabidopsis. Flavonoids are secondary metabolites derived from products of the phenylpropanoid biosynthetic pathway and the Krebs cycle. The final compounds include flavonols (colorless pigments), anthocyanins (pink and red pigments), and condensed tannins (brown pigments). The characterized enzymes are shown in boldface. The various mutants identified are noted adjacent to the step they affect, with putative regulatory loci given in parentheses. ban, banyuls; CHI, chalcone isomerase; CHS, chalcone synthase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; DFR, dihydroflavanol 4-reductase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; icx1, increased chalcone synthase expression1; LAR, leucoanthocyanidin reductase; LDOX, leucoanthocyanidin dioxygenase; PAL, phenylalanine ammonia-lyase; tt, transparent testa; ttg, transparent testa glabra.

Figure 2.

Phenotype of tt8 and Complemented Seeds. (A) Seeds of the deb122 mutant (right) of Arabidopsis compared with those of the wild-type genotype (left). Mutant and wild-type genotypes are both from the Wassilewskija-2 (Ws-2) ecotype. (B) T₂ progeny of a tt8-3 (deb122) homozygous plant transformed with the pBIB-Hyg T-DNA binary vector carrying the DEB122 genomic region. .

Figure 3.

Molecular Analysis of the TT8 Locus. (A) Diagram of the DEB122 region on BAC F17A8. The arrow indicates the orientation of the putative DEB122 protein. The primers deb122RB₁ and deb122LB₂, used to amplify the deb122 probe, are indicated. The two HindIII restriction sites were used to generate an 8-kb genomic fragment (pBIB-Hyg-8) for complementation assays. (B) Genomic organization of the TT8 gene (Ws-2 ecotype). The positions and relative sizes of the exons and introns of the genomic clone are indicated by black and white boxes, respectively. The respective positions of the translation start (ATG) and the translation stop (TAG) codons are shown. The solid bar represents the conserved bHLH region. The T-DNA (7 kb) is inserted into the second intron of the tt8-3 allele and causes a 29-bp deletion indicated by the striped box, whereas ethyl methanesulfonate mutagenesis causes a nucleotide transition in the sixth intron of the tt8-1 allele (shown by an arrow). The position of a transposon-like element (1.5 kb) in the Col-0 wild-type genome is shown by the dashed arrow. The primers used for molecular analyses are noted below the diagram. RB, right border; LB, left border.

Figure 4.

Nucleotide and Deduced Amino Acid Sequences of the TT8 cDNA. Uppercase letters indicate the extent of the full-length TT8 transcript (GenBank accession number AJ277509); the untranscribed 5′ region is in lowercase letters. The amino acid residues derived from the translation of this sequence are shown (one-letter code) below the corresponding codons. The functional translation stop site (TAG) is marked with an asterisk. Position 307 shows an amino acid polymorphism related to the Arabidopsis ecotype. A putative nuclear localization signal is shown in boldface above the bHLH domain, and the bHLH domain is boxed in black. The presumed TATA box is underlined, and the intron positions are shown by arrowheads. The arrows indicate the two polyadenylation sites found, and the corresponding putative polyadenylation signals are underlined by the dashed lines.

Figure 5.

Deduced TT8 Amino Acid Sequence and Comparison with bHLH-Related Protein Sequences. (A) Amino acid sequence comparison of the bHLH regions encoded by TT8 and several MYC-related proteins. Asterisks indicate amino acid residues found in MYC-related protein sequences from all eukaryotic organisms, and conserved amino acids are boxed in black. Dashes indicate gaps inserted to improve the alignment. Sequences shown are those from human c-MYC (GenBank accession number X00198), newt MRF-4 (X82836), Xenopus NeuroD (U28067), yeast CBF1 (M33620), maize IN1 (U57899), Antirrhinum DELILA (M84913), Arabidopsis RD22BP1 (AB000875), and Arabidopsis TT8 (this study). (B) Distance analysis of several plant bHLH-related factor sequences recovered by using a BLAST algorithm on the GenBank and EMBL databases. The following bHLH protein sequences were used to build the tree (accession numbers are in parentheses): maize Lc (M26227), maize R-S (X15806), maize B-Peru (X57276), maize IN1 (U57899), maize MYC7E (AF061107), rice Ra (U39860), pea PG1 (U18348), Gerbera MYC1 (AJ007709), bryophyte MYC-RP (AB024050), Antirrhinum DELILA (M84913), petunia Jaf13 (AF020545), and Arabidopsis AtMYC1 (D83511), Arabidopsis AtMYC146 (AF013465), Arabidopsis RD22BP1 (AB000875), and Arabidopsis TT8 (this study). Sequences were aligned using ClustalX and manually adjusted. For tree construction, only the N terminus region and the bHLH domain were used, as marked in (C). The consensus tree presented was obtained by neighbor-joining analysis, bootstrapped with 1000 iterations by ClustalX, and drawn with the TreeView program. Bootstrap values are indicated at each branchpoint; branches with a bootstrap score <850 were eliminated. Relative branch length (0.1) is indicated below the tree. Proteins in boldface have been reported to be involved in plant flavonoid metabolism. (C) Sequence comparison between TT8 and three other bHLH proteins involved in plant flavonoid pigmentation. Identical amino acids are boxed in black, and similar amino acids are boxed in gray. Brackets delimit the bHLH region. Sequences used are from maize IN1 (GenBank accession number U57899), maize B-Peru (X57276), Antirrhinum DELILA (M84913) and Arabidopsis TT8 (this study). Asterisks above the sequences indicate amino acid residues used to build the distance tree shown in (B). Dashes were introduced to optimize alignment.

Figure 6.

Analysis of tt8 Mutations. (A) Detection of TT8 transcripts in siliques of two tt8 mutants and their corresponding parental ecotype by RT-PCR. The full-length TT8 transcript was detected by ethidium bromide staining after 35 cycles of amplification. The expression of the EF1αA4 gene was used as a control. (B) Characterization of the donor and acceptor splicing sites of intron 6 in the wild type (En-2) and tt8-1 mutant. Brackets indicate correctly spliced sites; dots indicate an internal intron. Amino acid residues are shown (one-letter code) below the sequence. The asterisk indicates the mutated nucleotide in the tt8-1–derived sequence.

Figure 7.

Analysis of TT8 Expression during Plant Development. (A) TT8 transcripts were detected by quantitative RT-PCR in vegetative parts that included 4-day-old seedlings, rosette leaves, stems, and roots from 10-day-old plantlets and in reproductive organs that included buds, flowers, and developing seeds. Accumulation of the EF1αA4 transcript was used as an internal control. The PCR products were detected by DNA gel blot analysis after 21 amplification cycles. The blots were hybridized with the respective probes. (B) Comparison of TT8 expression pattern in reproductive organs with those of C4H, CHS, and DFR genes. PCR and hybridization assays were conducted exactly as those in (A).

Figure 8.

Expression of Flavonoid Biosynthetic Genes in Globular Embryo Stage Siliques. Siliques were obtained from wild-type genotype and three tt mutants: tt8-3, ttg1-1, and tt2-3. Expression of the different genes was monitored by quantitative RT-PCR.