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Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

Zabala G, Vodkin LO - PLoS ONE (2014)

Bottom Line: The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats.In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize.This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Sciences, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m) is homozygous for a mutable allele (r(m)) that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m) line had a 13 kb CACTA subfamily transposon insertion (designated TgmR*) at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3) to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-r(m) progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

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Features of the R2R3-MYB Transcription Factor Protein Sequence Encoded by the R Locus, Glyma09g36983.(A) The full length standard R allele. The extent of the imperfect R2 and R3 repeats is marked with the two dotted lines. Highlighted in black and gray are the conserved amino acids found in other higher plant MYB TFs of the R2R3 class (Lin-Wang et al, 2010). The amino acids highlighted black are best conserved. Aqua highlight is the [D/E]Lx2[R/K]x3Lx6Lx3R conserved amino acid signature functionally relevant in the MYB TF interaction with R/B-like bHLH proteins [23]. The conserved amino acids of this signature in the R locus encoded MYB are indicated in burgundy type. Green highlights the amino acids that are part of the motif found in AtMYB113 of subgroup 5 involved in anthocyanin regulation (activation) [10]. The divergent amino acids are indicated in red type. Highlighted fuchsia are four of the five amino acids constituting the transcriptional repression motif found in AtMYBs of subgroup 4 (AtMYB3, AtMYB4, AtMYB7, AtMYB32) [10]. (B) The truncated polypeptide resulting from the Exon-2 “C”-nt deletion in the defective r allele. It preserves only the R2 domain and 16 amino acids of the R3 domain.
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pone-0111959-g008: Features of the R2R3-MYB Transcription Factor Protein Sequence Encoded by the R Locus, Glyma09g36983.(A) The full length standard R allele. The extent of the imperfect R2 and R3 repeats is marked with the two dotted lines. Highlighted in black and gray are the conserved amino acids found in other higher plant MYB TFs of the R2R3 class (Lin-Wang et al, 2010). The amino acids highlighted black are best conserved. Aqua highlight is the [D/E]Lx2[R/K]x3Lx6Lx3R conserved amino acid signature functionally relevant in the MYB TF interaction with R/B-like bHLH proteins [23]. The conserved amino acids of this signature in the R locus encoded MYB are indicated in burgundy type. Green highlights the amino acids that are part of the motif found in AtMYB113 of subgroup 5 involved in anthocyanin regulation (activation) [10]. The divergent amino acids are indicated in red type. Highlighted fuchsia are four of the five amino acids constituting the transcriptional repression motif found in AtMYBs of subgroup 4 (AtMYB3, AtMYB4, AtMYB7, AtMYB32) [10]. (B) The truncated polypeptide resulting from the Exon-2 “C”-nt deletion in the defective r allele. It preserves only the R2 domain and 16 amino acids of the R3 domain.

Mentions: We searched for molecular domains and motifs in the R-encoded MYB protein sequence which may be predictors of its mechanistic role in the activation/repression of anthocyanin synthesis. The R locus MYB protein sequence contains at the N-terminus the R2R3 domains characteristic of the large R2R3-MYB gene family (125 genes) in Arabidopsis[10]. Figure 8 shows the conserved amino acids (highlighted in black and gray) in the R2 and R3 domains of the R gene MYB protein sequence.


Methylation affects transposition and splicing of a large CACTA transposon from a MYB transcription factor regulating anthocyanin synthase genes in soybean seed coats.

Zabala G, Vodkin LO - PLoS ONE (2014)

Features of the R2R3-MYB Transcription Factor Protein Sequence Encoded by the R Locus, Glyma09g36983.(A) The full length standard R allele. The extent of the imperfect R2 and R3 repeats is marked with the two dotted lines. Highlighted in black and gray are the conserved amino acids found in other higher plant MYB TFs of the R2R3 class (Lin-Wang et al, 2010). The amino acids highlighted black are best conserved. Aqua highlight is the [D/E]Lx2[R/K]x3Lx6Lx3R conserved amino acid signature functionally relevant in the MYB TF interaction with R/B-like bHLH proteins [23]. The conserved amino acids of this signature in the R locus encoded MYB are indicated in burgundy type. Green highlights the amino acids that are part of the motif found in AtMYB113 of subgroup 5 involved in anthocyanin regulation (activation) [10]. The divergent amino acids are indicated in red type. Highlighted fuchsia are four of the five amino acids constituting the transcriptional repression motif found in AtMYBs of subgroup 4 (AtMYB3, AtMYB4, AtMYB7, AtMYB32) [10]. (B) The truncated polypeptide resulting from the Exon-2 “C”-nt deletion in the defective r allele. It preserves only the R2 domain and 16 amino acids of the R3 domain.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219821&req=5

pone-0111959-g008: Features of the R2R3-MYB Transcription Factor Protein Sequence Encoded by the R Locus, Glyma09g36983.(A) The full length standard R allele. The extent of the imperfect R2 and R3 repeats is marked with the two dotted lines. Highlighted in black and gray are the conserved amino acids found in other higher plant MYB TFs of the R2R3 class (Lin-Wang et al, 2010). The amino acids highlighted black are best conserved. Aqua highlight is the [D/E]Lx2[R/K]x3Lx6Lx3R conserved amino acid signature functionally relevant in the MYB TF interaction with R/B-like bHLH proteins [23]. The conserved amino acids of this signature in the R locus encoded MYB are indicated in burgundy type. Green highlights the amino acids that are part of the motif found in AtMYB113 of subgroup 5 involved in anthocyanin regulation (activation) [10]. The divergent amino acids are indicated in red type. Highlighted fuchsia are four of the five amino acids constituting the transcriptional repression motif found in AtMYBs of subgroup 4 (AtMYB3, AtMYB4, AtMYB7, AtMYB32) [10]. (B) The truncated polypeptide resulting from the Exon-2 “C”-nt deletion in the defective r allele. It preserves only the R2 domain and 16 amino acids of the R3 domain.
Mentions: We searched for molecular domains and motifs in the R-encoded MYB protein sequence which may be predictors of its mechanistic role in the activation/repression of anthocyanin synthesis. The R locus MYB protein sequence contains at the N-terminus the R2R3 domains characteristic of the large R2R3-MYB gene family (125 genes) in Arabidopsis[10]. Figure 8 shows the conserved amino acids (highlighted in black and gray) in the R2 and R3 domains of the R gene MYB protein sequence.

Bottom Line: The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats.In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize.This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

View Article: PubMed Central - PubMed

Affiliation: Department of Crop Sciences, University of Illinois, Urbana, Illinois, United States of America.

ABSTRACT
We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-r(m) is homozygous for a mutable allele (r(m)) that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-r(m) line had a 13 kb CACTA subfamily transposon insertion (designated TgmR*) at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3) to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13 kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-r(m) progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock's Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element.

Show MeSH