Protein-coding genes are epigenetically regulated in Arabidopsis polyploids

Abstract

The fate of redundant genes resulting from genome duplication is poorly understood. Previous studies indicated that ribosomal RNA genes from one parental origin are epigenetically silenced during interspecific hybridization or polyploidization. Regulatory mechanisms for protein-coding genes in polyploid genomes are unknown, partly because of difficulty in studying expression patterns of homologous genes. Here we apply amplified fragment length polymorphism (AFLP)-cDNA display to perform a genome-wide screen for orthologous genes silenced in Arabidopsis suecica, an allotetraploid derived from Arabidopsis thaliana and Cardaminopsis arenosa. We identified ten genes that are silenced from either A. thaliana or C. arenosa origin in A. suecica and located in four of the five A. thaliana chromosomes. These genes represent a variety of RNA and predicted proteins including four transcription factors such as TCP3. The silenced genes in the vicinity of TCP3 are hypermethylated and reactivated by blocking DNA methylation, suggesting epigenetic regulation is involved in the expression of orthologous genes in polyploid genomes. Compared with classic genetic mutations, epigenetic regulation may be advantageous for selection and adaptation of polyploid species during evolution and development.

Figure 1

(a) AFLP-cDNA display of gene expression in diploid (2x) and autotetraploid (4x) Arabidopsis. The cDNA was synthesized with or without reverse transcriptase (+RT or −RT), respectively, and subjected to AFLP analysis. The primer pairs used in PCR were EcoRI-TA and MseI-CAT. Circles indicate the cDNA fragments present in both diploid and autotetraploid (open) and either the diploid or autotetraploid (filled). (b) AFLP-cDNA analysis in an allotetraploid, A. suecica (As), and its two diploid progenitors, A. thaliana (At) and C. arenosa (Ca). The primer pairs used in PCR were EcoRI-AC and MseI-CAC. Symbols indicate nonpolymorphic cDNA fragments (open circles), coexpression of orthologous alleles (arrows), and the cDNA alleles present only in one of the diploids and/or allotetraploid (open squares). Three alleles from one locus (filled square) were cloned and sequenced. (c) Sequencing gel results show a region of TCP3 from A. thaliana (AtTCP3), A. suecica (AsTCP3), and C. arenosa (CaTCP3). A base transition (G to A) was identified among the orthologous alleles (indicated by arrows). The size markers for a and b are shown to the left.

Figure 2

Differential expression patterns of parental genes in A. suecica. (a) Diagram of a TCP3-containing BAC clone (F12 M16). The BAC contains 30 predicted genes (gray boxes), some of which are not shown and indicated by double-slash lines. Five genes in the vicinity of TCP3 include RFP, HYP1, HYP2, TCP3, and MDH. The boxes indicate that expression patterns of parental genes in A. suecica are from C. arenosa only (black), A. thaliana only (open), or both (slash lines) origins (see b). (b) Parental gene expression patterns in A. suecica and its diploid progenitors. Agarose gels show RT-PCR results of four genes in A. thaliana (At), A. suecica (As), and C. arenosa (Ca). For every gene (e.g., RFP) the amplification was performed by addition of mock (lanes 2–4) or reverse transcriptase (lanes 5–10). The RT-PCR products of the RFP were then digested with XhoI (lanes 8–10). The products were resolved in a 1.5% agarose gel. The RT-PCR products amplified from the HYP1 (lanes 11–13), TCP3 (lanes 14–16), and MDH (lanes 17–19) were digested with BamHI, KpnI, and HindIII, respectively. The controls omitted for HYP1, TCP3, and MDH are RT-PCR without the reverse transcriptase and RT-PCR products without restriction digestion. EcoRI- and HindIII-digested lambda DNA is shown in lane 1 as size markers.

Figure 3

Silenced RFP is hypermethylated and demethylated by aza-dC in A. suecica. (a) Restriction map of the RFP genomic sequence (XbaI fragment) showing ten MspI/HpaII sites (labeled from 1 to 10) from the start codon to the end of the coding sequence. Five exons (open boxes) are joined by four introns (stippled lines). The probe used for DNA blot analysis is shown below the diagram. Sizes of the fragments resulting from complete digestion with XbaI (X) and MspI (X + M) or HapII (X + H) are shown. Lollipops indicate complete (filled), partial (half-filled), or no methylation (open) at the sites shown. (b) Genomic DNA isolated from young leaves of A. suecica (As) and its diploid progenitors, A. thaliana (At) and C. arenosa (Ca), was digested with XbaI (lanes 1–3). Each sample was divided equally into three parts; two of the samples were digested again with another restriction enzyme, MspI or HapII. The DNA samples were then separated in agarose gel, blotted, and hybridized with the probe indicated above. A. suecica has two XbaI fragments (lane 2), one from A. thaliana (lane 1) and another from arenosa (lane 3). Eight of nine HpaII sites are partially methylated in A. thaliana (compare lanes 4 and 7), although completely methylated in A. suecica (compare lanes 8 and 7). Only a few HpaII/MspI sites were detected in the C. arenosa gene (lanes 6 and 9). (c) Demethylation of the AtRFP genomic sequence after aza-dC treatment. Genomic DNA was isolated from seedlings of the A. suecica treated with (+) or without (−) addition of aza-dC (10 mg/L), a chemical inhibitor of DNA methyltransferases. The DNA was digested by XbaI and MspI or HpaII, as mentioned, and subjected to DNA blot analysis by using the same probe.

Figure 4

Reactivation of the silenced RFP and TCP3 in A. suecica. (a) RNA was isolated from young leaves of A. thaliana (At), C. arenosa (Ca), and A. suecica treated with (As+) or without (As) addition of 10 mg/l aza-dC. The mRNA was subjected to RT-PCR and cleaved amplified polymorphic sequence (CAPS) analyses (28). Negative controls (−RT, lanes 2–4) indicate PCR amplification is completely dependent on reverse transcriptase. The amplified products (lanes 6–9) were digested by XhoI (lanes 10–13). The thaliana-like RFP transcripts were absent in A. suecica before aza-dC treatment (lane 11), but reactivated after the treatment (lane 12). (b) The silenced C. arenosa-TCP3 is reactivated by aza-dC. The RT-PCR products were amplified by using TCP3-specific primers (lanes 14–17) and digested with KpnI (lanes 18–21). The C. arenosa-like TCP3 transcripts were absent in A. suecica before aza-dC treatment (lane 19), but reactivated after the treatment (lane 20). EcoRI- and HindIII-digested lambda DNA is shown lanes 1 and 22 as a size marker.