Abstract
Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.
Highlights
Knowledge of the RNA silencing pathway in plants is advanced, but relatively few examples exist of regulation of a specific plant phenotype by naturally occurring variation in the pathway
We investigated the presence of chalcone synthase (CHS)-related short interfering RNAs (siRNAs) species in seed coats of the nonpigmented (Richland, I), and hilumpigmented isoline (Williams, ii) along with their corresponding mutant allele lines (T157, i and Williams 55, i) (Table 1) using RNA gel blotting
CHS7 was chosen as the probe since the nearly identical CHS7 and CHS8 genes are downregulated by the silencing I locus (Senda et al, 2004; Tuteja et al, 2004)
Summary
Knowledge of the RNA silencing pathway in plants ( known as RNA interference) is advanced (reviewed in Baulcombe, 2004; Matzke and Matzke, 2004; Zamore and Haley, 2005; Chapman and Carrington, 2007; Eamens et al, 2008; Ramachandran and Chen, 2008; Carthew and Sontheimer, 2009), but relatively few examples exist of regulation of a specific plant phenotype by naturally occurring variation in the pathway. The soybean (Glycine max) I (inhibitor) locus, an unusual cluster arrangement of chalcone synthase (CHS) genes that inhibits seed coat pigmentation, is one such example of a silencing locus (Todd and Vodkin, 1996; Tuteja et al, 2004) mediated through posttranscriptional RNA silencing that can be suppressed by a viral silencing suppressor protein (Senda et al, 2004). The homozygous recessive i allele allows for pigment production and accumulation over the entire epidermal layer of the seed coat. Most cultivated soybean varieties have been selected for a yellow, nonpigmented seed coat (homozygous I or ii alleles) to mitigate the undesirable effects of the black or brown anthocyanin pigments on protein and oil extractions during processing of soybean products (Palmer et al, 2004)
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