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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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Phenotypes of Seeds Coats at Late Developmental Stages for Three Soybean Isolines.Differential anthocyanin expression in the seed coats of three soybean isolines with variants of the R locus alleles. RM55-rm with variegated black-brown seed coat; RM30-R* a stable revertant black-seeded isoline derived from RM55-rm; and RM38-r a brown seeded isoline used as the recurrent parent. See Table 1 for more information. Left, green seed of approximately 300–400 mg fresh weight; middle, dessicating seed of approximately 300–400 mg fresh weight; right, mature dry seed.
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pone-0111959-g001: Phenotypes of Seeds Coats at Late Developmental Stages for Three Soybean Isolines.Differential anthocyanin expression in the seed coats of three soybean isolines with variants of the R locus alleles. RM55-rm with variegated black-brown seed coat; RM30-R* a stable revertant black-seeded isoline derived from RM55-rm; and RM38-r a brown seeded isoline used as the recurrent parent. See Table 1 for more information. Left, green seed of approximately 300–400 mg fresh weight; middle, dessicating seed of approximately 300–400 mg fresh weight; right, mature dry seed.

Mentions: The rm allele of the R locus conditions a variegated or mutable distribution of black spots or concentric rings of black pigment superimposed on an otherwise brown seed coat (Figure 1). This phenotype is sometimes referred to as a striped phenotype. The rm allele present in a plant introduction line was backcrossed for five generations into a brown seeded Clark line with homozygous i, r, T genotype to create isoline L72-2040 with i rm, T genotype and released in 1972 by soybean breeder R.L. Bernard of the USDA Agricultural Research Service. We discovered that the rm variegated seed of L72-2040 demonstrated somatic and germinal instability to yield fully black seed [6]. One of these lines, here named as RM30, is shown in Figure 1 and the gene symbol R* is used to differentiate this stable revertant allele, derived from the rm line, from the standard R allele that specifics black seed coats. The mutable line is here referred to as RM55-rm and the brown isoline into which the rm allele was backcrossed is RM38-r (Figure 1).


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)

Phenotypes of Seeds Coats at Late Developmental Stages for Three Soybean Isolines.Differential anthocyanin expression in the seed coats of three soybean isolines with variants of the R locus alleles. RM55-rm with variegated black-brown seed coat; RM30-R* a stable revertant black-seeded isoline derived from RM55-rm; and RM38-r a brown seeded isoline used as the recurrent parent. See Table 1 for more information. Left, green seed of approximately 300–400 mg fresh weight; middle, dessicating seed of approximately 300–400 mg fresh weight; right, mature dry seed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111959-g001: Phenotypes of Seeds Coats at Late Developmental Stages for Three Soybean Isolines.Differential anthocyanin expression in the seed coats of three soybean isolines with variants of the R locus alleles. RM55-rm with variegated black-brown seed coat; RM30-R* a stable revertant black-seeded isoline derived from RM55-rm; and RM38-r a brown seeded isoline used as the recurrent parent. See Table 1 for more information. Left, green seed of approximately 300–400 mg fresh weight; middle, dessicating seed of approximately 300–400 mg fresh weight; right, mature dry seed.
Mentions: The rm allele of the R locus conditions a variegated or mutable distribution of black spots or concentric rings of black pigment superimposed on an otherwise brown seed coat (Figure 1). This phenotype is sometimes referred to as a striped phenotype. The rm allele present in a plant introduction line was backcrossed for five generations into a brown seeded Clark line with homozygous i, r, T genotype to create isoline L72-2040 with i rm, T genotype and released in 1972 by soybean breeder R.L. Bernard of the USDA Agricultural Research Service. We discovered that the rm variegated seed of L72-2040 demonstrated somatic and germinal instability to yield fully black seed [6]. One of these lines, here named as RM30, is shown in Figure 1 and the gene symbol R* is used to differentiate this stable revertant allele, derived from the rm line, from the standard R allele that specifics black seed coats. The mutable line is here referred to as RM55-rm and the brown isoline into which the rm allele was backcrossed is RM38-r (Figure 1).

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