Gene dosage and stochastic effects determine the severity and direction of uniparental ribosomal RNA gene silencing (nucleolar dominance) in Arabidopsis allopolyploids

Abstract

Nucleolar dominance is an epigenetic phenomenon in which one parental set of ribosomal RNA (rRNA) genes is silenced in an interspecific hybrid. In natural Arabidopsis suecica, an allotetraploid (amphidiploid) hybrid of Arabidopsis thaliana and Cardaminopsis arenosa, the A. thaliana rRNA genes are repressed. Interestingly, A. thaliana rRNA gene silencing is variable in synthetic Arabidopsis suecica F1 hybrids. Two generations are needed for A. thaliana rRNA genes to be silenced in all lines, revealing a species-biased direction but stochastic onset to nucleolar dominance. Backcrossing synthetic A. suecica to tetraploid A. thaliana yielded progeny with active A. thaliana rRNA genes and, in some cases, silenced C. arenosa rRNA genes, showing that the direction of dominance can be switched. The hypothesis that naturally dominant rRNA genes have a superior binding affinity for a limiting transcription factor is inconsistent with dominance switching. Inactivation of a species-specific transcription factor is argued against by showing that A. thaliana and C. arenosa rRNA genes can be expressed transiently in the other species. Transfected A. thaliana genes are also active in A. suecica protoplasts in which chromosomal A. thaliana genes are repressed. Collectively, these data suggest that nucleolar dominance is a chromosomal phenomenon that results in coordinate or cooperative silencing of rRNA genes.

Figure 1

Nucleolar dominance in A. suecica. (A) Organization of the intergenic spacers separating 25S and 18S rRNA coding sequences of adjacent rRNA genes. A. thaliana intergenic spacers are longer and contain repetitive elements smaller than those located in C. arenosa spacers. (B) Ethidium bromide-stained agarose gel of PCR products using the primers depicted in A and A. suecica, A. thaliana, and C. arenosa genomic DNA. HindIII-digested λDNA served as size markers in lane 1. The PCR reaction in lane 5 was performed with both primers but no genomic DNA. (C) Detection of rRNA transcripts by using the S1 nuclease protection assay. A. thaliana, C. arenosa, and natural A. suecica rRNA transcripts were detected with C. arenosa- (lanes 3–5) or A. thaliana-specific (lanes 6–8) DNA probes. Dideoxynucleotide sequencing reactions served as size markers in lanes 1 and 2.

Figure 2

Creation of synthetic A. suecica allotetraploids and backcross progeny. A. thaliana is typically diploid, with a haploid complement of five chromosomes. C. arenosa is a natural tetraploid with a haploid complement of eight chromosomes. Tetraploid A. thaliana was used as the maternal parent in a cross with C. arenosa, yielding SAS plants. SAS F₂ progeny were obtained from self-pollinated flowers. Alternatively, flowers were emasculated and manually pollinated with tetraploid A. thaliana or C. arenosa pollen. Thus progeny with 3:1, 2:2 (F₂), or 1:3 A. thaliana to C. arenosa genome complements were obtained from the same mother plant.

Figure 3

Variable severity of nucleolar dominance in SAS F₁ hybrids. Total RNA from SAS, A. thaliana, and C. arenosa plants (lanes 7 and 8) was assayed by using S1 nuclease protection with A. thaliana- (lanes 3, 4, 8–10) or C. arenosa-specific probes (lanes 5–7, 11, 12). DNA sequencing reactions (lanes 1 and 2) served as size markers.

Figure 4

Two generations are needed for establishment of nucleolar dominance in some SAS lines. (A) SAS-1, in which A. thaliana and C. arenosa rRNA genes were codominant, was self-pollinated to generate F₂ plants. rRNA transcripts in six F₂ siblings and C. arenosa and A. thaliana controls (lanes 7, 8) were detected using C. arenosa- (lanes 1–7) or A. thaliana-specific (lanes 8–14) S1 probes. (B) F₂ progeny of SAS-2 were analyzed in the same way.

Figure 5

The direction of nucleolar dominance can be switched. SAS-2, which displayed complete dominance of C. arenosa over A. thaliana rRNA genes (see Fig. 3) was the maternal parent for backcrosses to tetraploid A. thaliana or C. arenosa. (A) Five backcross sibs from each cross were assayed for rRNA gene expression using A. thaliana- (Top) or C. arenosa-specific (Bottom) S1 probes. A. thaliana or C. arenosa controls are in lane 1 of the relevant autoradiograms. (B) Southern blot analysis of BssH II-digested DNA of A. thaliana (lane 1), C. arenosa (lane 2), SAS-2 (lane 3), or the backcross sibs (lanes 4–8) tested in A. The filter was first hybridized to an A. thaliana-specific rRNA gene intergenic spacer probe (Top), then was stripped and reprobed (49) with a similar C. arenosa probe (Bottom). The former corresponded to −2,590 to +6 of plasmid pAt1 5′Δ−2,590/3′Δ+6 (27); the latter spanned −2,316 to +33 of pCa1.

Figure 6

A. thaliana and C. arenosa rRNA minigenes are transcribed in the other species. A. thaliana (At), C. arenosa (Ca) or A. suecica (As) protoplasts were transfected with 50 pmols of pBluescript plasmid containing C. arenosa rRNA gene promoter sequences (pCa; sequences −265 to ≈+1,700) or one of two A. thaliana minigenes, pAt or pAt*. Constructs pAt and pAt* correspond to minigenes pAt1 5′Δ−520 (sequences −520 to +92) and pAt1 5′Δ−2,590/3′Δ+6 (−2,590 to +6), respectively (27). Transcripts were detected with minigene-specific S1 probes. Transcripts in lanes 3 and 4 derive from an experiment in which 50 pmols each of pAt and pCa were cotransfected and half of the isolated RNA was hybridized with the At (lane 3) or Ca (lane 4) probe. Data in lanes 1–4 and 5–9 are derived from the same exposure of a single autoradiogram. Note that no transcripts are detected in protoplasts transfected only with pBluescript (P) DNA (lanes 7–9).

Figure 7

Derepression of A. thaliana rRNA genes in natural A. suecica using 5-aza-2′ deoxycytidine (aza-dC). Approximately 30 A. suecica seeds were surface-sterilized and germinated on medium containing 0, 10, or 25 mg/L, as described previously (33). (A) Total RNA from 3-week-old plantlets was purified and analyzed by S1 nuclease protection with A. thaliana- (lanes 2–5) or C. arenosa-specific (lanes 1, 6–8) probes. C. arenosa and A. thaliana control reactions are in lanes 1 and 2. (B) Southern blot analysis of DNA from control and treated plants following digestion with HpaII (lanes 1–3) or MspI (lanes 4–6) and hybridization to an A. thaliana probe spanning the ≈2-kb region from −365 to the beginning of the 18S rRNA coding sequences. This probe detects both A. thaliana and C. arenosa rRNA genes.