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Tissue specificity of chalcone synthase short interfering RNAs (CHS siRNAs) that control seed color

ABSTRACT: We previously showed CHS siRNAs levels to be high in immature seed coats of seed with dominant alleles of the I locus that controls seed color (Tuteja et al., Plant Cell 2009; Cho et al., Plos One, 2013) and to be much lower in several other tissues sampled (Zabala et al., BMC Plant Biology 2012). Here, we examined 15 additional small RNA populations by high-throughput sequencing of developing seed coats of various stages from cultivars with the dominant I and i-i alleles that specify yellow seed coats or from spontaneous mutations to pigmented seed coats. The CHS siRNAs were tissue specific and found at high levels of up to 5700 normalized RPMs (reads per millions) in immature seed coats but were less than 6 RPMs in libraries from 21 non-seed coat samples including the embryonic axis, different stages of seedlings cotyledons, vegetative buds, and shoot tips, illustrating the tightly controlled tissue-specific expression of the CHS siRNAs in seed coats.

ORGANISM(S): Glycine max

PROVIDER: GSE239484 | GEO | 2025/07/31

REPOSITORIES: GEO

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

Project description:We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype. In order to compare the population of chalcone synthase related small RNAs, we sequenced 3 to 6 million small RNAs using the Illumina Genome Analyzer from the following four soybean cultivars and tissues with specific genotypes at the I locus: Richland immature seed coats (homozygous for the dominant I allele that specifies yellow seed coat); Williams immature seed coats (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum) Williams (i-i/i-i yellow) immature cotyledons (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum); Williams 55 immature seed coats (a Williams isogenic line homozygous for the recessive i allele that specifics pigmented seed coats. All seed coats and cotyledons were dissected from green stage immature seeds within the fresh weight range of 50-75 mg.

2010-05-13 | E-GEOD-21825 | biostudies-arrayexpress

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

2010-05-14 | GSE21825 | GEO

Mutations in Argonaute5 Illuminate Epistatic Interactions of the K1 and I Loci Leading to Saddle Seed Color Patterns in Glycine max

Project description:The soybean (Glycine max) seed coat has distinctive, genetically programmed patterns of pigmentation and the recessive k1 mutation can epistatically overcome the dominant I and i-i alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that CHS siRNA levels cause the patterns produced by the i-i and i-k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but different genotypes (homozygous i-k K1 versus homozygous i-i k1). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting AGO5, thus establishing Glyma.11G190900 as the K1 locus. Non-functional AGO5 from k1 alleles leads to altered distributions of CHS siRNAs, thus explaining how the k1 mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant I or i-i alleles.

2017-03-30 | GSE43347 | GEO

Mutations in Argonaute5 Illuminate Epistatic Interactions of the K1 and I Loci Leading to Saddle Seed Color Patterns in Glycine max

2017-03-30 | GSE89126 | GEO

Multiple Origins of Chalcone Synthase Short Interfering RNAs in Glycine max Revealed by Genome Assemblies of Mutant Alleles

Project description:With long-read assemblies, we delineate three origins of short interfering RNAs for chalcone synthase (CHS siRNAs) in Glycine max. Mutations from ii (yellow seed coat with black hilum) to i (fully pigmented) lack either a single antisense CHS1 gene by deletion or the partial 5’ subtilisin fragment-CHS1 gene region by a large inversion mutation. On the other hand, we show that the dominant I allele, with fully yellow seed coats, arose from the i black, wild soybean genome by an inverted duplication that places a partial 5’ DnaJ fragment next to slightly truncated CHS genes. In a direct I to i mutation, the two CHS genes are further truncated, destroying their capacity to produce siRNAs. The two-color saddle pattern of the ik allele arose by an inverted duplication event that places a partial 5’ P450 fragment in front of two inverted repeat CHS genes. Despite the importance of the three different 5’ promoters that abut inverted repeat CHS genes as drivers of precursor expression, the profiles of cognate gene family members for subtilisin, DnaJ, and P450 are not always limited to the seed coat as tightly as CHS siRNAs, implying that transcriptional or processing events for CHS dsRNA or siRNAs differ in other tissues.

2025-07-31 | GSE239923 | GEO

The Transition from Primary siRNAs to Amplified Secondary siRNAs that Regulate Chalcone Synthase During Development of Glycine max Seed Coat

Project description:The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.

2014-02-13 | GSE43348 | GEO

The Transition from Primary siRNAs to Amplified Secondary siRNAs that Regulate Chalcone Synthase During Development of Glycine max Seed Coat

2014-02-13 | E-GEOD-43348 | biostudies-arrayexpress

Tissue specific expression of chalcone synthase siRNAs

Project description:In a previous study, seed coat and cotyledon tissues of Williams, Richland and T157 soybean lines were investigated to show tissue specificity of CHS siRNA expression (Tuteja et al., 2009). Here, we investigated more tissues such as leaf, root and germinating cotyledon to ascertain the tissue specificity of CHS siRNAs in Williams. Data from multiple small RNA libraries were sequenced deeply by the Illumina high-throughput sequencing technology. The total numbers of small RNA reads were from three million to thirty million, providing sufficient data to show the tissue specificity of CHS siRNA.

2014-02-13 | GSE49708 | GEO

Tissue specific expression of chalcone synthase siRNAs

2014-02-13 | E-GEOD-49708 | biostudies-arrayexpress

Small RNA from Arabidopsis thaliana with RNA silencing directed to Chalcone Synthase (At5G13930) by a hairpin RNA, a 21-nt artificial miRNA and a 22-nt artificial miRNA in the pap1-D over-expressing background

Project description:22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger trans-acting phased small interfering RNA (tphasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA) mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA DEPENDENT RNA POLYMERASE6 mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families. sRNA sequencing from aerial tissues of 3-week old plants grown on MS media

2013-10-14 | E-GEOD-49792 | biostudies-arrayexpress

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