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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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Differential Expression of the MYB Transcription Factor Encoded by the R Locus in Developing Seed Coats of a Stable Black-Seeded Soybean Revertant Line RM30-R* and a Brown-Seeded Line RM38-r.(A) Soybean seed developmental stages for the RM38-r (brown), RM55-rm (variegated) and RM30-R* stable black revertant. Encircled in red are the five developmental stages (in mg seed fresh weight) chosen for the RNA-Seq analysis. See Table S1 for the sequence read counts of each sample which ranged from from 30 to 77 million. (B) Expression of Glyma09g36983 in RPKMs plotted against the five stages of seed development in mg seed fresh weight as shown in (A) above. 1: 100–200 mg, 2: 200–300 mg, 3: 300–400 mg, 4: 400–500 mg and 5: 300–400 mg as the seeds enter desiccation. The solid red line represents transcripts derived from seed coats of the RM38-r brown-seeded line without the TgmR* but with a “C”-nt deletion in Exon2. The blue line represent the expression in seed coats of the RM30-R* black revertant line with the TgmR* insertion in Intron2.
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pone-0111959-g004: Differential Expression of the MYB Transcription Factor Encoded by the R Locus in Developing Seed Coats of a Stable Black-Seeded Soybean Revertant Line RM30-R* and a Brown-Seeded Line RM38-r.(A) Soybean seed developmental stages for the RM38-r (brown), RM55-rm (variegated) and RM30-R* stable black revertant. Encircled in red are the five developmental stages (in mg seed fresh weight) chosen for the RNA-Seq analysis. See Table S1 for the sequence read counts of each sample which ranged from from 30 to 77 million. (B) Expression of Glyma09g36983 in RPKMs plotted against the five stages of seed development in mg seed fresh weight as shown in (A) above. 1: 100–200 mg, 2: 200–300 mg, 3: 300–400 mg, 4: 400–500 mg and 5: 300–400 mg as the seeds enter desiccation. The solid red line represents transcripts derived from seed coats of the RM38-r brown-seeded line without the TgmR* but with a “C”-nt deletion in Exon2. The blue line represent the expression in seed coats of the RM30-R* black revertant line with the TgmR* insertion in Intron2.

Mentions: We compared the expression Glyma09g36983 MYB transcription factor in the seed coats of the black revertant RM30-R* soybean line to that in the defective brown RM38-r soybean line at various stages of seed development. Total RNA was extracted from seed coats at five stages of seed development (Figure 4A) following a modified method that prevents RNA adhesion to the proanthocyanidins (tannins) that accumulate in the vacuoles of pigmented seed coats [17], [18]. The five stages of seed development were chosen based on the fresh weight of entire seeds: 100–200, 200–300, 300–400 and 400–500 mg with green cotyledons, and a later stage in which seed desiccation had initiated judging by the yellowing of cotyledons and a lower fresh weight of 300–400 mg.


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)

Differential Expression of the MYB Transcription Factor Encoded by the R Locus in Developing Seed Coats of a Stable Black-Seeded Soybean Revertant Line RM30-R* and a Brown-Seeded Line RM38-r.(A) Soybean seed developmental stages for the RM38-r (brown), RM55-rm (variegated) and RM30-R* stable black revertant. Encircled in red are the five developmental stages (in mg seed fresh weight) chosen for the RNA-Seq analysis. See Table S1 for the sequence read counts of each sample which ranged from from 30 to 77 million. (B) Expression of Glyma09g36983 in RPKMs plotted against the five stages of seed development in mg seed fresh weight as shown in (A) above. 1: 100–200 mg, 2: 200–300 mg, 3: 300–400 mg, 4: 400–500 mg and 5: 300–400 mg as the seeds enter desiccation. The solid red line represents transcripts derived from seed coats of the RM38-r brown-seeded line without the TgmR* but with a “C”-nt deletion in Exon2. The blue line represent the expression in seed coats of the RM30-R* black revertant line with the TgmR* insertion in Intron2.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111959-g004: Differential Expression of the MYB Transcription Factor Encoded by the R Locus in Developing Seed Coats of a Stable Black-Seeded Soybean Revertant Line RM30-R* and a Brown-Seeded Line RM38-r.(A) Soybean seed developmental stages for the RM38-r (brown), RM55-rm (variegated) and RM30-R* stable black revertant. Encircled in red are the five developmental stages (in mg seed fresh weight) chosen for the RNA-Seq analysis. See Table S1 for the sequence read counts of each sample which ranged from from 30 to 77 million. (B) Expression of Glyma09g36983 in RPKMs plotted against the five stages of seed development in mg seed fresh weight as shown in (A) above. 1: 100–200 mg, 2: 200–300 mg, 3: 300–400 mg, 4: 400–500 mg and 5: 300–400 mg as the seeds enter desiccation. The solid red line represents transcripts derived from seed coats of the RM38-r brown-seeded line without the TgmR* but with a “C”-nt deletion in Exon2. The blue line represent the expression in seed coats of the RM30-R* black revertant line with the TgmR* insertion in Intron2.
Mentions: We compared the expression Glyma09g36983 MYB transcription factor in the seed coats of the black revertant RM30-R* soybean line to that in the defective brown RM38-r soybean line at various stages of seed development. Total RNA was extracted from seed coats at five stages of seed development (Figure 4A) following a modified method that prevents RNA adhesion to the proanthocyanidins (tannins) that accumulate in the vacuoles of pigmented seed coats [17], [18]. The five stages of seed development were chosen based on the fresh weight of entire seeds: 100–200, 200–300, 300–400 and 400–500 mg with green cotyledons, and a later stage in which seed desiccation had initiated judging by the yellowing of cotyledons and a lower fresh weight of 300–400 mg.

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