Tissue-Specific siRNAs That Silence CHS Genes in Soybean

Chalcone synthase (CHS) is required for the biosynthesis of anthocyanin pigments that give color to various plant tissues, such as the flower and seed coat. The silencing of CHS genes produces a highly visible phenotype, lack of color in the seed coat or flower, that facilitated the discovery of gene silencing in eukaryotes (Napoli et al., 1990) and continues to provide a useful system for investigating the mechanistic basis and regulation of the phenomenon. In soybean (Glycine max), two naturally occurring dominant alleles of the I locus (I and ii ) silence nine CHS genes to inhibit pigmentation in the seed coat, resulting in a colorless or light-yellow seed coat. By contrast, the homozygous recessive i allele allows for expression of CHS genes, resulting in pigment production and a dark brown or black color in the seed coat. Most cultivated soybean varieties have been selected for homozygous I or ii alleles to mitigate undesirable effects of anthocyanin pigments on protein and oil extractions. These varieties continue to express CHS genes in other plant tissues, suggesting that there is tissue specificity to silencing in the seed coat. It has been presumed that silencing is due to the production of small interfering RNA (siRNA) at the CHS locus, which in I and ii genotypes is a complex locus containing a series of duplicated and inverted CHS genes (Clough et al., 2004; Senda et al., 2004; Tuteja et al., 2004; Tuteja and Vodkin, 2008). However, the mechanistic basis and tissue specificity of this silencing have not been fully characterized. In this issue, Tuteja et al. (pages 3063– 3077) investigate the CHS-derived small RNA populations in several soybean varieties using RNA gel blotting and highthroughput sequencing of small RNAs. They show that CHS siRNAs accumulate in the yellow seed coats of plants carrying dominant I or ii alleles but not in pigmented seed coats with a homozygous recessive i genotype. Furthermore, in the I and ii genotypes, CHS siRNAs are restricted to the seed coat and absent from cotyledons of developing seed. Therefore, the dominant I and ii alleles confer a highly tissue-specific accumulation of CHS siRNAs, leading to silencing of the genes only in the seed coat. The authors show that the population of small RNAs in the seed coat of the yellow ii genotype maps primarily to the coding regions (both sense and antisense strands) of CHS and is predominantly 21 nucleotides in length (see figure). They conclude that silencing from the complex CHS loci in soybean I and ii varieties occurs because these loci contain clusters of CHS sequences in inverse orientation (some of which are deleted in the recessive i genotypes), which generate RNA transcripts that could fold and create aberrant doublestranded RNA (dsRNA). The latter are diced to generate primary siRNAs that target other CHS family member genes. The size and sequence distribution of the CHSsiRNA is consistent with the hypothesis that aberrant dsRNA are synthesized and processed by an RNA-dependent RNA polymerase, dicer-like, and argonaute-like effector complex that amplifies the silencing signal by cleavage of these dsRNAs into phased 21to 22-nucleotide secondary siRNAs (Chapman and Carrington, 2007). This naturally occurring instance of gene silencing is analogous in some ways to cosuppression of CHS in petunia, in which plants transformed with extra copies of CHS produce aberrant dsRNA that leads to silencing of the endogenous CHS genes in floral tissue. De Paoli et al. (2009) recently provided evidence that the induction of CHS siRNAs is causally related to the cosuppression of CHS in petunia that gives rise to loss of flower pigmentation. In petunia, there are at least six CHS genes (CHS-A, -B, -D, -F, -J, and -H), but only