Expression analysis of Arabidopsis XH/XS-domain proteins indicates overlapping and distinct functions for members of this gene family

Abstract

RNA-directed DNA methylation (RdDM) is essential for de novo DNA methylation in higher plants, and recent reports established novel elements of this silencing pathway in the model organism Arabidopsis thaliana. Involved in de novo DNA methylation 2 (IDN2) and the closely related factor of DNA methylation (FDM) are members of a plant-specific family of dsRNA-binding proteins characterized by conserved XH/XS domains and implicated in the regulation of RdDM at chromatin targets. Genetic analyses have suggested redundant as well as non-overlapping activities for different members of the gene family. However, detailed insights into the function of XH/XS-domain proteins are still elusive. By the generation and analysis of higher-order mutant combinations affected in IDN2 and further members of the gene family, we have provided additional evidence for their redundant activity. Distinct roles for members of the XH/XS-domain gene family were indicated by differences in their expression and subcellular localization. Fluorescent protein-tagged FDM genes were expressed either in nuclei or in the cytoplasm, suggestive of activities of XH/XS-domain proteins in association with chromatin as well as outside the nuclear compartment. In addition, we observed altered location of a functional FDM1-VENUS reporter from the nucleus into the cytoplasm under conditions when availability of further FDM proteins was limited. This is suggestive of a mechanism by which redistribution of XH/XS-domain proteins could compensate for the loss of closely related proteins.

Keywords: Arabidopsis; RdDM.; gene family; protein localization.

Fig. 1.

Analysis of DNA methylation and gene silencing in idn2/fdm mutants. (A) Southern blot performed with HpaII-digested (left lanes) and HaeIII-digested (right lanes) genomic DNA probed with the labelled 5S rRNA gene. Genotypes are indicated on top. (B) Southern blot performed with HaeIII-digested genomic DNA probed with labelled AtMU1 DNA. Genotypes are indicated on top. (C) Southern blot performed with HpaII-cut (left lanes) and MspI-cut (right lanes) genomic DNA that was probed for MEA-ISR. Genotypes are indicated on top. (D) TS-GUS activity in 14-d-old plantlets as determined by the 4-MUG assay. Activities are plotted as fold induction of activity found in the TS-GUS controls (given a value of 1). Standard deviations are indicated (n=4); the asterisk indicates a significant difference in reporter activity between TS-GUS and idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2 TS-GUS (t-test, P<0.05). (E) TS-GUS reporter activity in 14-d-old TS-GUS (left) and idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2 TS-GUS (right) plantlets. Blue staining indicates release of gene silencing.

Fig. 2.

Expression analysis of XH/XS-domain genes in Arabidopsis. (A) RT-PCR performed with cDNA generated from seedling (sdl.), root, true leaf (leaf), and flower mRNA. UQB5 transcript levels were used for standardization. (B–D) GUS staining of FDM1p::GUS (B), FDM2p::GUS (C), and FDM5p::GUS (D) in roots at 8 days after germination (DAG). FDM2p::GUS is predominantly active in the vasculature and emerging lateral roots (C). (E–G) GUS staining of FDM1p::GUS (B), FDM2p::GUS (C), and FDM5p::GUS (D) seedlings at 6 DAG. (H–K) GUS staining of FDM1p::GUS (H), FDM2p::GUS (J) and FDM5p::GUS (K) at 15 DAG. (L–N) GUS-stained flowers of FDM1p::GUS (L), FDM2p::GUS (M) and FDM5p::GUS (N) from 28-d-old plants. Bars, 75 μm (B, C); 5mm (E–G); 2.5mm (H–K); 1mm (L–N).

Fig. 3.

Subcellular localization of FDM–VENUS reporters (green, yellow) in live Col-0 root meristems (top panels) and in root meristem epidermis cells at higher magnification (bottom panels). (A) FDM1p::FDM1:VENUS. (B) FDM5p::FDM5:VENUS. (C) FDM1p::FDM5:VENUS. (D) FDM5p::FDM1:VENUS. (E) RP40p::FDM1:VENUS. (F) RP40p::FDM5:VENUS. Roots were counterstained with propidium iodide (100 µg ml^–1, red) to visualize cell boundaries. Bars, 50 μm (top panels); 10 μm (bottom panels).

Fig. 4.

Functional analysis of FDM–VENUS reporter proteins. (A–D) TS-GUS staining in 10-d-old TS-GUS (A), idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2 TS-GUS (B), idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2 TS-GUS RP40p::FDM1:VENUS (C), and idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2 TS-GUS RP40p::FDM5:VENUS (D). (E) TS-GUS activity in 14-d-old plantlets as determined by a 4-MUG assay. Activities are plotted as fold induction of activity found in the TS-GUS controls (given a value of 1). Two independent experiments are shown and standard deviation is indicated. Asterisks and brackets highlight significant differences in TS-GUS activity between samples (t-test; P<0.05).

Fig. 5.

Subcellular localization of FDM–VENUS reporter proteins (yellow) in fixed root meristem cells of idn2/fdm mutants. (A) RP40p::FDM1:VENUS (left panels) and RP40p::FDM5:VENUS (right panels) in fdm1-1. Note the accumulation of FDM1–VENUS reporter signals in the nucleolar cavity, whilst weak signals only are found in the surrounding dense fibrillar zone. (B) RP40p::FDM1:VENUS (left panels) and RP40p::FDM5:VENUS (right panels) in fdm5-2. (C) RP40p::FDM1:VENUS (left panels) and RP40p::FDM5:VENUS (right panels) in idn2-3 fdm1-1 idp2-1 fdm3-2 fdm4-2 fdm5-2. Seedlings were fixed in formaldehyde (3.7%, v/v) and counterstained with DAPI (0.5ng ml^–1, blue) to visualize nuclei. Bars,10 μm.