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-rm is homozygous for a mutable allele (rm) 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-rm 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-rm 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.
Highlights
Anthocyanins are end products of three branches of the flavonoid pathway with important functions in plant defense against pathogens and protection from UV light
The translated MYB protein drives an increase in transcript accumulation of the anthocyanidin synthase 2 (ANS2)/3, UFGT2 and AOMT genes whose products function in the late steps of anthocyanin synthesis, in production of the cyanidin-3-glycoside that accounts for the black pigmentation of the seed coats
The observed higher level of transcripts found for the defective r allele in RM38-r seed coats may translate into truncated peptides that are non-functional and do not activate the transcription of the late anthocyanin pathway genes resulting in brown seeds which reflects accumulation of only proanthocyanidins
Summary
Anthocyanins are end products of three branches of the flavonoid pathway with important functions in plant defense against pathogens and protection from UV light. Because of their antioxidant properties, seeds and vegetables with anthocyanin pigments have added nutritional and health value. Understanding the regulation and expression of all regulatory genes in the anthocyanin metabolic pathway in each major crop plant species is of significance as a model system for gene expression and for improving agronomic and nutritional properties. We have identified the molecular basis of some of the classical loci leading to anthocyanin production in soybean (Glycine max) seed and plant parts. Since CHS is the first committed enzyme in the anthocyanin pathway, the I genotypes result in yellow seed coats.
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