Endogenous, tissue-specific short interfering RNAs silence the chalcone synthase gene family in glycine max seed coats

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.

Figure 1.

CHS-Derived siRNAs in Seed Coats of Soybeans with Silencing Genotypes, Williams (iⁱ) and Richland (I). LMW RNA samples (75 μg) were fractionated in a 15% polyacrylamide gel and probed with an antisense CHS7 riboprobe transcribed from a full-length CHS7 cDNA. Radiolabeled LMW RNAs from both the yellow seed coat varieties Richland (I, yellow) and Williams (iⁱ, yellow seed coat with pigmented hilum) indicate the accumulation of CHS siRNA. By contrast, the LMW RNA fractions from the corresponding mutant isolines T157 (i) and Williams 55 (i) with pigmented seed coats lack CHS siRNA. Radiolabeled Decade markers (20 to 30 nucleotides) are shown at left and right.

Figure 2.

Cotyledons of Seeds with Silencing I and iⁱ Genotypes Do Not Accumulate CHS siRNA. LMW RNA fractions (75 μg) from seed coats and cotyledons of the soybean isogenic lines Richland (I, yellow seed coat) and T157 (i, pigmented seed coat) were separated on 15% polyacrylamide gels, and the resulting RNA gel blots were probed with an antisense CHS7 riboprobe transcribed from a full-length CHS7 cDNA. CHS siRNAs were detected only in the seed coats of Richland (I), the cultivar with the yellow seed coats (top panel). The bottom panel shows small RNAs stained with ethidium bromide.

Figure 3.

CHS siRNAs Accumulate in Seed Coats but Not in the Vegetative Tissues of Yellow Seeded Lines. LMW RNA fractions (75 μg) were separated on 15% polyacrylamide gels and the RNA gel blots probed with an antisense CHS7 riboprobe transcribed from a full-length CHS7 cDNA. CHS siRNAs were detected only in the seed coats of the yellow seeded cultivars Williams (iⁱ; [A]) with the hilum pigmented yellow seed coats (lane 1, top panel) or) Richland (I; [B]) with yellow seed (lane 1, top panel) but not in cotyledons, leaves, and roots of either soybean line or their respective pigmented isolines Williams 55 (i; [A]) or T157 (I; [B]). Radiolabeled Decade markers (20 to 30 nucleotides) are shown at right. Lower panel shows hybridization of the same LMW RNA fractions to a 5S rRNA probe to show equal LMW RNA sample loading.

Figure 4.

Schematic Diagram Mapping the Total Count of Small RNAs from the Seed Coat versus the Cotyledon Libraries Both Made from the Silencing Williams Genotype (iⁱ, Yellow Seeds) to Their Locations on Five BAC Clones Containing Members of the CHS Gene Gamily. Total numbers of small RNA sequence reads related to the five BACs (77G7a, 56G2, 5A23, 28017, and C7C24) were obtained from the nearly three million sequence reads obtained by Illumina from seed coat (SC top line) or cotyledon (COT bottom line) libraries of Williams (iⁱ) yellow seeds. Closed arrows represent open reading frames in the indicated direction of transcription. Dark closed arrows indicate CHS genes, and light arrows represent other annotated genes as shown by Tuteja and Vodkin (2008). Annotations are shown only for CHS genes and for some of the transposon related open reading frames denoted by pink open arrows. The size of BACs in base pairs and the number of genes (excluding transposons) are given to the right of each BAC. See Methods for the BLAST criteria.

Figure 5.

Diagram Representing Abundance and Alignments of CHS siRNAs with Sequence Signatures Identical to CHS7 or CHS4 Genes. CHS siRNAs from the seed coat library of the yellow seed Williams (iⁱ) having 100% match to the CHS7 (or CHS4) gene sequences were mapped based on their alignments to specific locations. The intron and two exons of CHS7 and CHS4 are depicted, and the orientation of transcription is indicated with an arrowhead. The colored segments represent the number of occurrences in the library and the location of their alignment with CHS7 or CHS4 sequences. The “x” denotes one siRNA signature spanning the intron. Those aligning to the sense strand are above the gene, and those aligning to the antisense strand are denoted below the gene. Number of occurrences: 50 to 100 (light blue), 100 to 250 (dark blue), 250 to 500 (green), 500 to 750 (orange), and 750 to 1000 (red). The boxed-in region highlights the most targeted portion of exon 2.

Figure 6.

Size Distributions of CHS siRNAs for Each CHS Gene in a CHS-Silenced Seed Coat Library. CHS siRNAs selected from the seed coat of yellow seed, Williams iⁱ, small RNA library were filtered to identify those with 100% identity to individual CHS genes. The nine CHS-siRNA subgroups were distributed according to the size of individual sequence signatures and plotted in two graphs. (A) The number of unique signatures of a given size for each CHS gene. (B) The same as in (A) but multiplied by the number of occurrences of each CHS-siRNA signature. The result for each CHS gene is color coded as indicated.

Figure 7.

A Schematic Illustrating the Role of CHS Gene Clusters in Generation of CHS siRNAs in the Silencing iⁱ Allele and Its Comparison to the Recessive i Mutation. Seed phenotypes are indicated for W = Williams (iⁱ, hilum-only pigmented seed coat) and the isogenic mutant line W55 = Williams 55 (i, black seed coat). The presence of an exact, base-by-base duplication of the 10.91-kb CHS clusters A and B at the I locus as revealed by BAC sequencing of the yellow genotype (iⁱ) is diagrammed, as is the deletion in the i mutation. Marked by green Xs, the deletion encompasses regions flanking CHS cluster B and extends into the promoter region, including the HindIII (H3) site of CHS4 in cluster A. RFLP analysis also shows absence of the 2.3-kb HindIII fragment corresponding to CHS4 genes in the pigmented genotype (i). (Summarized from Todd and Vodkin, 1996; Tuteja et al., 2004). The molecular events supported by the CHS-siRNA data presented in this report are diagrammed. A dsRNA generated from the inverted CHS repeats in the seed coat is cleaved into primary siRNAs representing both strands that are amplified by RdRP to generate secondary CHS siRNAs capable of downregulating all members of the CHS gene family, including the more distantly related CHS7 and CHS8 (denoted in red). These two genes are highly expressed in the pigmented seed coats in which CHS siRNA production has been abolished by the deletion in the mutant i allele (W55). Production of the primary CHS siRNAs is tissue specific, found only in the seed coats and not in other tissues of the yellow seeded (iⁱ) genotype.