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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.

Show MeSH
Differential Expression of Genes that Function in the Early Steps of the Anthocyanin and Proanthocyanidin Biosynthetic Pathways Between the Black-Seeded RM30-R* and Brown-Seeded RM38-r Lines.Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in Figure 4 for RM30-R* where the R* allele is interrupted by the 13 kb TgmR* insertion (profile in blue) and the RM38-r line where the r allele is not interrupted by TgmR* but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as follows: CHS7 (Glyma01g43880.1); CHS8 (Glyma11g01350.1); CHI1A (Glyma20g38560.1); CHI2 (Glyma20g38580.1); F3′H (Glyma06g21920.1); F3′5′H (Glyma13g04210.1); F3H (Glyma02g05450.1); DFR1 (Glyma14g07940.1); DFR2 (Glyma17g37060.1. See Figure 7 for the pathway abbreviations.
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pone-0111959-g005: Differential Expression of Genes that Function in the Early Steps of the Anthocyanin and Proanthocyanidin Biosynthetic Pathways Between the Black-Seeded RM30-R* and Brown-Seeded RM38-r Lines.Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in Figure 4 for RM30-R* where the R* allele is interrupted by the 13 kb TgmR* insertion (profile in blue) and the RM38-r line where the r allele is not interrupted by TgmR* but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as follows: CHS7 (Glyma01g43880.1); CHS8 (Glyma11g01350.1); CHI1A (Glyma20g38560.1); CHI2 (Glyma20g38580.1); F3′H (Glyma06g21920.1); F3′5′H (Glyma13g04210.1); F3H (Glyma02g05450.1); DFR1 (Glyma14g07940.1); DFR2 (Glyma17g37060.1. See Figure 7 for the pathway abbreviations.

Mentions: Contrary to the difference observed for the expression of the MYB gene in the two lines in Figure 4B, where higher levels were measured in the brown RM38-r seed coats, all other pathway genes analyzed had higher RPKM expression values in the black RM30-R* line at the late stages of seed development (Figures 5 and 6, Table 2). These increases occurred at the developmental time when the R locus gene, Glyma09g36983, is activated. One exception is the ANR genes for which the low level of expression at the late stages of seed development was maintained in the seed coats of the black RM30-R* line as well as in the brown RM38-r line (Table 2). This is consistent with the function of this gene's product in directing the synthesis of proanthocyanidins now in competition for its substrate that is being diverted towards anthocyanin synthesis in the black seed coats at the late stages of seed development as shown in Figure 7.


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 Genes that Function in the Early Steps of the Anthocyanin and Proanthocyanidin Biosynthetic Pathways Between the Black-Seeded RM30-R* and Brown-Seeded RM38-r Lines.Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in Figure 4 for RM30-R* where the R* allele is interrupted by the 13 kb TgmR* insertion (profile in blue) and the RM38-r line where the r allele is not interrupted by TgmR* but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as follows: CHS7 (Glyma01g43880.1); CHS8 (Glyma11g01350.1); CHI1A (Glyma20g38560.1); CHI2 (Glyma20g38580.1); F3′H (Glyma06g21920.1); F3′5′H (Glyma13g04210.1); F3H (Glyma02g05450.1); DFR1 (Glyma14g07940.1); DFR2 (Glyma17g37060.1. See Figure 7 for the pathway abbreviations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111959-g005: Differential Expression of Genes that Function in the Early Steps of the Anthocyanin and Proanthocyanidin Biosynthetic Pathways Between the Black-Seeded RM30-R* and Brown-Seeded RM38-r Lines.Transcript levels are in RPKMs plotted against the same five stages of seed coat development as shown in Figure 4 for RM30-R* where the R* allele is interrupted by the 13 kb TgmR* insertion (profile in blue) and the RM38-r line where the r allele is not interrupted by TgmR* but has a “C”-nt deletion in Exon2 (red profile). Graphs have different scales. The Glyma models for the indicated genes are as follows: CHS7 (Glyma01g43880.1); CHS8 (Glyma11g01350.1); CHI1A (Glyma20g38560.1); CHI2 (Glyma20g38580.1); F3′H (Glyma06g21920.1); F3′5′H (Glyma13g04210.1); F3H (Glyma02g05450.1); DFR1 (Glyma14g07940.1); DFR2 (Glyma17g37060.1. See Figure 7 for the pathway abbreviations.
Mentions: Contrary to the difference observed for the expression of the MYB gene in the two lines in Figure 4B, where higher levels were measured in the brown RM38-r seed coats, all other pathway genes analyzed had higher RPKM expression values in the black RM30-R* line at the late stages of seed development (Figures 5 and 6, Table 2). These increases occurred at the developmental time when the R locus gene, Glyma09g36983, is activated. One exception is the ANR genes for which the low level of expression at the late stages of seed development was maintained in the seed coats of the black RM30-R* line as well as in the brown RM38-r line (Table 2). This is consistent with the function of this gene's product in directing the synthesis of proanthocyanidins now in competition for its substrate that is being diverted towards anthocyanin synthesis in the black seed coats at the late stages of seed development as shown in Figure 7.

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