Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport

Abstract

We have developed a high-throughput T-DNA insertional mutagenesis program in tomato using activation tagging to identify genes that regulate metabolic pathways. One of the activation-tagged insertion lines (ant1) showed intense purple pigmentation from the very early stage of shoot formation in culture, reflecting activation of the biosynthetic pathway leading to anthocyanin accumulation. The purple coloration resulted from the overexpression of a gene that encodes a MYB transcription factor. Vegetative tissues of ant1 plants displayed intense purple color, and the fruit showed purple spotting on the epidermis and pericarp. The gene-to-trait relationship of ant1 was confirmed by the overexpression of ANT1 in transgenic tomato and in tobacco under the control of a constitutive promoter. Suppression subtractive hybridization and RNA hybridization analysis of the purple tomato plants indicated that the overexpression of ANT1 caused the upregulation of genes that encode proteins in both the early and later steps of anthocyanidin biosynthesis as well as genes involved in the glycosylation and transport of anthocyanins into the vacuole.

Figure 1.

Fruit Color and Shape Mutants from Activation-Tagging Transgenic Lines. (A) Wild-type fruit. (B) Dark green fruit. (C) Golden yellow fruit. (D) Orange fruit. (E) Salmon pink fruit. (F) Cross-sections of wild-type and salmon pink fruit. (G) Pear shape. (H) Persimmon shape. (I) Radish shape. (J) Teardrop shape. (K) Yeast-budding-type mutants. All fruit variants were observed in the T0 generation except the salmon color mutant ([E] and [F]), which was observed in T1.

Figure 2.

ant1 Mutant. (A) Wild-type and mutant plants of T1. (B) Wild-type flower. (C) Mutant flower of T0. (D) Surface of a green fruit of the ant1 mutant from T0 at ×66 magnification.

Figure 3.

Structure and Expression of the Predicted Gene in the ant1 Mutant. (A) Plasmid rescued from the anthocyanin mutant, and the predicted gene structure of ANT1 activated by 4× 35S enhancer. (B) RT-PCR analysis of ANT1 expression. WT, wild type.

Figure 4.

Alignment of Predicted Protein Sequences of ANT1 and Related MYB Factors. (A) Amino acid alignment of the R2R3 MYB domain of functionally characterized plant proteins involved in the regulation of flavonoid biosynthesis. The tomato MYB factor identified in this study (LeANT1) is compared with petunia AN2, Arabidopsis PAP1, Arabidopsis PAP2, Arabidopsis TT2, maize Pl, and maize C1. Residues that are identical to residues in the tomato ANT1 sequence are shaded. The positions of the conserved W/I residues implicated in DNA binding are indicated below the alignment with asterisks. (B) Amino acid sequence alignment of the full-length tomato ANT1 and petunia AN2 proteins showing identical (asterisks) and similar (dashes) residues (CLUSTAL W, version 1.4: open gap penalty of 10.0; extended gap penalty of 0.1; similarity matrix of blosum62).

Figure 5.

ANT1 Recapitulated Tomato Plants AX, BK, and AW in the Greenhouse. The degree of purpleness in flowers and fruit was correlated directly to the increased levels of transcript and anthocyanin pigment, respectively, in the seedling analyses of the various events.

Figure 6.

RT-PCR of ANT1 Transgenic Plants of Micro-Tom. Plants 1 to 6 were transgenic plants. The Actin gene was used as a positive control. ANT1 was overexpressed in all plants that were purple in appearance (lanes 1, 2, 4, 5, and 6). Lane 3 contained RNA from a transgenic plant that was positive for ANT1 DNA (data not shown) but showed no purple color. WT, wild type.

Figure 7.

Recapitulation of Tobacco with ANT1 Gene. At left, wild-type and ANT1 transgenic plants of tobacco cv Wisconsin. At right, RT-PCR data. All of the plants of ANT1 transgenic lines of tobacco were positive for the transcript (lanes 1 to 5). WT, wild type.

Figure 8.

Average Anthocyanin Content of Transgenic and Wild-Type Micro-Tom Plants (3-Week-Old Whole Seedlings). Error bars indicate standard errors for n = 4. F.Wt, fresh weight.

Figure 9.

ANT1 Expression Levels in Transgenic Tomato Lines AW, BK, and AX. (A) Equal RNA loading. (B) and (C) Blots probed with radiolabeled DNA fragments corresponding to ANT1 or MTP4 encoding GST. WT, wild type.

Figure 10.

SMART cDNA Gel Blot Hybridization. SMART cDNA from 3-week-old seedlings of wild-type Micro-Tom (lanes 1) or ANT1 transgenic Micro-Tom, event BK (lanes 2), was separated, transferred, and probed with a radiolabeled DNA fragment corresponding to actin and each labeled gene. In all cases, the transcripts that were upregulated in the ANT1 transgenic tomato were undetectable in the untransformed control. Note the putative alternate polyadenylated forms of tomato DFR transcript. The numbers (in kb) indicate the estimated molecular masses of the hybridizing bands.

Figure 11.

Amino Acid Sequence Alignments of CHI from Various Species. Sequences are from tomato MTP96 (this study), Arabidopsis At3g6317, citrus CHI, rice CHI, alfalfa CHI A and CHI B, and petunia CHI1 and CHI2. Identical amino acid residues conserved across species are indicated below the alignment with asterisks, and similar amino acids are indicated with dashes.

Figure 12.

Amino Acid Sequence Alignments of the Putative Anthocyanin Permease from Various Species. Sequences are from tomato MTP77 (this study), Arabidopsis TT12, the At4g00350 gene product, and the At4g25640 gene product. Identical amino acid residues conserved across species are indicated below the alignment with asterisks, and similar amino acids are indicated with dashes.