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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 RNA-Seq Reads from Seed Coats of the RM30-R* and RM38-r When Aligned to the Transcript Sequence of Glyma09g36983.The pair of graphs in each row represent the distribution of non-normalized RNAseq reads that align to the 714 bp transcript sequence from the Glyma09g36983 gene at each of the five stages of seed coat development indicated in the margin to the right (described in Figure 4). The plots in the left column represent the distributions of sequence reads derived from the seed coats of the RM30-R* that is interrupted by the TgmR* transposon and the plots in the right column represent the RNAseq reads derived from the RM38-r line with the uninterrupted R gene but containing the “C”-nt deletion at 222 bp of the transcript. The numbers of RNAseq reads matching a given position of the R gene transcript are plotted against the position along the reference Glyma09g36983 transcript sequence containing the three exons and no introns. No expression was detected in the early 100–200 mg seed developmental stage in the RM30-R* line. The red arrow points to the location of the r allele “C”-nt deletion at 222 bp of the transcript.
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pone-0111959-g009: Distribution of RNA-Seq Reads from Seed Coats of the RM30-R* and RM38-r When Aligned to the Transcript Sequence of Glyma09g36983.The pair of graphs in each row represent the distribution of non-normalized RNAseq reads that align to the 714 bp transcript sequence from the Glyma09g36983 gene at each of the five stages of seed coat development indicated in the margin to the right (described in Figure 4). The plots in the left column represent the distributions of sequence reads derived from the seed coats of the RM30-R* that is interrupted by the TgmR* transposon and the plots in the right column represent the RNAseq reads derived from the RM38-r line with the uninterrupted R gene but containing the “C”-nt deletion at 222 bp of the transcript. The numbers of RNAseq reads matching a given position of the R gene transcript are plotted against the position along the reference Glyma09g36983 transcript sequence containing the three exons and no introns. No expression was detected in the early 100–200 mg seed developmental stage in the RM30-R* line. The red arrow points to the location of the r allele “C”-nt deletion at 222 bp of the transcript.

Mentions: We inspected the alignment of the RNA-Seq reads from the Rm30-R* and RM38-r lines at all five developmental stages (Table 2) against the 714-nt (3-exon gene model) transcript sequence with Bowtie 1. The results presented in Figure 9 show clearly the numbers of sequence reads and their alignments with a total coverage of the Glyma09g36983 gene transcript sequence in both lines. The expression level of the R gene is low in black and brown seed coats with the only significant difference manifested in seed coats of the 400–500 mg seeds, with the brown seed coats accumulating slightly more than double the level in the black seed coats (Figure 9) as previously indicated in Table 2 with the RM30-R*/RM38-r ratio of 0.49. As stated earlier, these alignment results also show that the Glyma09g36983 gene is transcribed all the way to the 3′end in the RM30-R* black seed coats (Figure 9) despite the large transposon insertion at 110 nt from the beginning of Intron2. These data indicate that despite a very low level of transcripts, they are cleanly processed at the intron exon boundaries.


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 RNA-Seq Reads from Seed Coats of the RM30-R* and RM38-r When Aligned to the Transcript Sequence of Glyma09g36983.The pair of graphs in each row represent the distribution of non-normalized RNAseq reads that align to the 714 bp transcript sequence from the Glyma09g36983 gene at each of the five stages of seed coat development indicated in the margin to the right (described in Figure 4). The plots in the left column represent the distributions of sequence reads derived from the seed coats of the RM30-R* that is interrupted by the TgmR* transposon and the plots in the right column represent the RNAseq reads derived from the RM38-r line with the uninterrupted R gene but containing the “C”-nt deletion at 222 bp of the transcript. The numbers of RNAseq reads matching a given position of the R gene transcript are plotted against the position along the reference Glyma09g36983 transcript sequence containing the three exons and no introns. No expression was detected in the early 100–200 mg seed developmental stage in the RM30-R* line. The red arrow points to the location of the r allele “C”-nt deletion at 222 bp of the transcript.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111959-g009: Distribution of RNA-Seq Reads from Seed Coats of the RM30-R* and RM38-r When Aligned to the Transcript Sequence of Glyma09g36983.The pair of graphs in each row represent the distribution of non-normalized RNAseq reads that align to the 714 bp transcript sequence from the Glyma09g36983 gene at each of the five stages of seed coat development indicated in the margin to the right (described in Figure 4). The plots in the left column represent the distributions of sequence reads derived from the seed coats of the RM30-R* that is interrupted by the TgmR* transposon and the plots in the right column represent the RNAseq reads derived from the RM38-r line with the uninterrupted R gene but containing the “C”-nt deletion at 222 bp of the transcript. The numbers of RNAseq reads matching a given position of the R gene transcript are plotted against the position along the reference Glyma09g36983 transcript sequence containing the three exons and no introns. No expression was detected in the early 100–200 mg seed developmental stage in the RM30-R* line. The red arrow points to the location of the r allele “C”-nt deletion at 222 bp of the transcript.
Mentions: We inspected the alignment of the RNA-Seq reads from the Rm30-R* and RM38-r lines at all five developmental stages (Table 2) against the 714-nt (3-exon gene model) transcript sequence with Bowtie 1. The results presented in Figure 9 show clearly the numbers of sequence reads and their alignments with a total coverage of the Glyma09g36983 gene transcript sequence in both lines. The expression level of the R gene is low in black and brown seed coats with the only significant difference manifested in seed coats of the 400–500 mg seeds, with the brown seed coats accumulating slightly more than double the level in the black seed coats (Figure 9) as previously indicated in Table 2 with the RM30-R*/RM38-r ratio of 0.49. As stated earlier, these alignment results also show that the Glyma09g36983 gene is transcribed all the way to the 3′end in the RM30-R* black seed coats (Figure 9) despite the large transposon insertion at 110 nt from the beginning of Intron2. These data indicate that despite a very low level of transcripts, they are cleanly processed at the intron exon boundaries.

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