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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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Distribution of Methylation in the TgmR* Transposon (A) and in the Upstream (B) and Downstream Regions (C) of the R locus in the Black-seeded RM30-R* and the Mutable RM55-rm Isolines, and of the UC44 line with a Standard R Allele.All graphs were created by the sequence alignment tool of the Integrative Genomics Viewer (IGV) browser. (A) TgmR* methylation regions. The 13 bp CACTA inverted repeat ends and subterminal repeats are marked IR. Lower levels of methylated sequences (marked by red arrows) are found aligning to the subterminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped mutable RM55-rm line. The black-seeded RM30-R* line containing the TgmR* insertion and the black-seeded UC44-R, line which lacks the TgmR* insertion, present nearly identical patterns of methylation at those three differentiated areas of the TgmR* element. (B) Upstream regions and (C) downstream regions of the TgmR* insertion site in the R gene. A few methylation differences appear between the three lines in the upstream and downstream regions, but the most distinct are those for UC44-R (Williams self-black seed) which may represent varietal differences. Gene represented by green arrows are: A (Glyma09g36941); B (Glyma09g36950); C (Glyma09g36966); D (Glyma09g36983); E (Glyma09g37000) and F (Glyma09g37010). The upstream and downstream schematics are not drawn to scale.
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pone-0111959-g010: Distribution of Methylation in the TgmR* Transposon (A) and in the Upstream (B) and Downstream Regions (C) of the R locus in the Black-seeded RM30-R* and the Mutable RM55-rm Isolines, and of the UC44 line with a Standard R Allele.All graphs were created by the sequence alignment tool of the Integrative Genomics Viewer (IGV) browser. (A) TgmR* methylation regions. The 13 bp CACTA inverted repeat ends and subterminal repeats are marked IR. Lower levels of methylated sequences (marked by red arrows) are found aligning to the subterminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped mutable RM55-rm line. The black-seeded RM30-R* line containing the TgmR* insertion and the black-seeded UC44-R, line which lacks the TgmR* insertion, present nearly identical patterns of methylation at those three differentiated areas of the TgmR* element. (B) Upstream regions and (C) downstream regions of the TgmR* insertion site in the R gene. A few methylation differences appear between the three lines in the upstream and downstream regions, but the most distinct are those for UC44-R (Williams self-black seed) which may represent varietal differences. Gene represented by green arrows are: A (Glyma09g36941); B (Glyma09g36950); C (Glyma09g36966); D (Glyma09g36983); E (Glyma09g37000) and F (Glyma09g37010). The upstream and downstream schematics are not drawn to scale.

Mentions: The distribution of differentially methylated TgmR* regions in the black-seeded RM30-R* and the mutable RM55-rm soybean isolines is shown in Figure 10A as visualized by the sequence alignment tool of the Integrative genomics viewer (IGV) browser http://www.broadinstitute.org/software/igv/home[31]. Significantly lower levels of methylated sequences appear to align to the terminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped black/brown-seeded mutable RM55-rm line. The black-seeded line UC44-R line lacking the TgmR* insertion in R presents nearly identical pattern and level of methylation in the three areas of the TgmR* as in the RM30-R* line.


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)

Distribution of Methylation in the TgmR* Transposon (A) and in the Upstream (B) and Downstream Regions (C) of the R locus in the Black-seeded RM30-R* and the Mutable RM55-rm Isolines, and of the UC44 line with a Standard R Allele.All graphs were created by the sequence alignment tool of the Integrative Genomics Viewer (IGV) browser. (A) TgmR* methylation regions. The 13 bp CACTA inverted repeat ends and subterminal repeats are marked IR. Lower levels of methylated sequences (marked by red arrows) are found aligning to the subterminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped mutable RM55-rm line. The black-seeded RM30-R* line containing the TgmR* insertion and the black-seeded UC44-R, line which lacks the TgmR* insertion, present nearly identical patterns of methylation at those three differentiated areas of the TgmR* element. (B) Upstream regions and (C) downstream regions of the TgmR* insertion site in the R gene. A few methylation differences appear between the three lines in the upstream and downstream regions, but the most distinct are those for UC44-R (Williams self-black seed) which may represent varietal differences. Gene represented by green arrows are: A (Glyma09g36941); B (Glyma09g36950); C (Glyma09g36966); D (Glyma09g36983); E (Glyma09g37000) and F (Glyma09g37010). The upstream and downstream schematics are not drawn to scale.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111959-g010: Distribution of Methylation in the TgmR* Transposon (A) and in the Upstream (B) and Downstream Regions (C) of the R locus in the Black-seeded RM30-R* and the Mutable RM55-rm Isolines, and of the UC44 line with a Standard R Allele.All graphs were created by the sequence alignment tool of the Integrative Genomics Viewer (IGV) browser. (A) TgmR* methylation regions. The 13 bp CACTA inverted repeat ends and subterminal repeats are marked IR. Lower levels of methylated sequences (marked by red arrows) are found aligning to the subterminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped mutable RM55-rm line. The black-seeded RM30-R* line containing the TgmR* insertion and the black-seeded UC44-R, line which lacks the TgmR* insertion, present nearly identical patterns of methylation at those three differentiated areas of the TgmR* element. (B) Upstream regions and (C) downstream regions of the TgmR* insertion site in the R gene. A few methylation differences appear between the three lines in the upstream and downstream regions, but the most distinct are those for UC44-R (Williams self-black seed) which may represent varietal differences. Gene represented by green arrows are: A (Glyma09g36941); B (Glyma09g36950); C (Glyma09g36966); D (Glyma09g36983); E (Glyma09g37000) and F (Glyma09g37010). The upstream and downstream schematics are not drawn to scale.
Mentions: The distribution of differentially methylated TgmR* regions in the black-seeded RM30-R* and the mutable RM55-rm soybean isolines is shown in Figure 10A as visualized by the sequence alignment tool of the Integrative genomics viewer (IGV) browser http://www.broadinstitute.org/software/igv/home[31]. Significantly lower levels of methylated sequences appear to align to the terminal repeats of the TgmR* element and the 3′-end of the predicted ORF2 transcript in the striped black/brown-seeded mutable RM55-rm line. The black-seeded line UC44-R line lacking the TgmR* insertion in R presents nearly identical pattern and level of methylation in the three areas of the TgmR* as in the RM30-R* line.

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