A distant coilin homologue is required for the formation of cajal bodies in Arabidopsis

Abstract

Cajal bodies (CBs) are subnuclear bodies that are widespread in eukaryotes, being found in mammals, many other vertebrates and in all plant species so far examined. They are mobile structures, moving, fusing, and budding within the nucleus. Here we describe a screen for Arabidopsis mutants with altered CBs and describe mutants that have smaller Cajal bodies (ncb-2, ncb-3), lack them altogether (ncb-1), have increased numbers of CBs (pcb) or have flattened CBs (ccb). We have identified the gene affected in the ncb mutants as a distant homolog of the vertebrate gene that encodes coilin (At1g13030) and have termed the resulting protein Atcoilin. A T-DNA insertional mutant in this gene (ncb-4) also lacks Cajal bodies. Overexpression of Atcoilin cDNA in ncb-1 restores Cajal bodies, which recruit U2B'' as in the wild type, but which are, however, much larger than in the wild type. Thus we have shown that At1g13030 is required for Cajal body formation in Arabidopsis, and we hypothesize that the level of its expression is correlated with Cajal body size. The Atcoilin gene is unaffected in pcb and ccb, suggesting that other genes can also affect CBs.

Figure 1.

Confocal images of wild-type (1214), ncb-1 and ncb-2 root epidermal tissue. All lines express U2B″:GFP. (A) In the parental line all nuclei show clear, bright nuclear foci corresponding to CBs, as well as a lower level of nucleoplasmic labeling. The label is mostly excluded from the nucleolus. (B) In ncb-1 no CBs are visible and the nucleoplasmic labeling is generally brighter than in the parental line. Labeling is often visible in the central regions of the nucleoli. (C) In ncb-2, many nuclei show CB foci, but these are significantly smaller than in the parental line. The nucleoplasm and the central region of the nucleoli are labeled. Bar, 10 μm.

Figure 2.

Visualization of U2B″:GFP labeling in four other mutants identified. In each case a typical hypotocotyl cell is shown, imaged by wide-field CCD microscopy followed by image deconvolution. (A–E) Single z sections; (F) a z projection. (A) Wild type (1214) shows a single, clear CB. ncb-3 (B) and A3572 (C) both have significantly smaller CBs, similar to ncb-2. (D) ccb displays flattened CCBs around the nucleolar periphery. (E) pcb has an increased number of CBs. (F) Projection of the entire nucleus shown in E to clearly show the increased number of CBs. (There are no additional CBs besides those shown for the other lines.) Note that, in contrast to the rapidly dividing root cell nuclei, shown in Figure 1, the nuclei of many elongated plant cells are themselves elongated. Bar, 5 μm.

Figure 3.

Visualization of U2B″:GFP labeling in different cell types of wild type (1214; left-hand column), ncb-1(middle column), and ncb-2 (right-hand column). (A–C) Hypocotyl cells. (D–F) Leaf epidermal cells. (G–I) Root epidermal cells. (J–L) Root hair cells. (M–O) Trichome cells. In all cell types examined, whereas the parental line shows bright, clear CBs, in ncb-1 CBs are not visible, and in ncb-2 CBs are either much reduced in size or absent. All images were collected by wide-field epifluorescence using a CCD camera. The arrows indicate the nucleoli. Bar, 5 μm.

Figure 4.

Transmission electron microscopy and anti-fibrillarin labeling of root nuclei. (A) EM of parental (1214) line showing a clear CB (arrow) next to the nucleolus. (B) ncb-1 never shows visible CBs in the EM. N, nucleus; No, nucleolus; Het, heterochromatin. Bar, 1 μm in A and B. (C) Parental (1214) line also shows clear CBs in every nucleus after immunofluorescence labeling with anti-fibrillarin (arrows). (D) In ncb-1 no CBs are visible. (z projections of deconvoluted wide-field image stacks). Bar, 5 μm in C and D.

Figure 5.

Comparison of Atcoilin with vertebrate coilins. (A) The entire sequence of Atcoilin, with regions of significant homology with vertebrate coilin highlighted in green. (B) Alignment of Atcoilin residues 1–100 and vertebrate coilins. (C) Alignment of Atcoilin residues 409–511 and vertebrate coilins.

Figure 6.

Diagram showing the location of the base changes and transcript analysis of ncb-1 and ncb-2. (A) In ncb-1 a change from G to A has occurred at the 5′ end of exon 12. In ncb-2 a change from G to A has occurred at the 3′ end of exon 9. (B) RT-PCR of wild type (1214), ncb-1 and ncb-2 mRNA. Lane 1, markers. Lane 2, wild type with primers F9 and R12 shows a single band of the predicted size. Lane 3, ncb-1 with primers F9 and R12 shows two bands, one of about the same size as the wild type and one significantly larger. Lane 4, wild type with primers F9 and R10 shows a single band of the predicted size. Lane 5, ncb-2 with primers F9 and R10 shows two band, one of about the same size as the wild type and one significantly larger. (C) Diagram of the transcripts of wild-type cDNA from At1g13030 and the two transcripts from each of ncb-1 and ncb-2 and of the predicted polypeptides from each transcript. In the predicted proteins, the wild-type residues are shown in green, with the aberrant residues in red.

Figure 7.

Immunofluorescence labeling of CBs in parental (1214) line and T-DNA insertional mutant GABI-Kat line 233H02 in At1g13030. Root tissue was labeled with antibody 4G3, specific for U2B″. Images collected by wide-field epifluorescence using a CCD camera. (A and B) In the parental line CBs are clearly seen. (C and D) In the insertion line no CBs are seen. Arrows indicate the nucleoli. Bar, 5 μm.

Figure 8.

CBs in an ncb-1 line in which mRFP:Atcoilin is over expressed using the 35S promoter. Single optical sections from 3D CCD data after deconvolution. The different cell types correspond to those shown for the parental (1214) line, ncb-1 and ncb-2 in Figure 3. Left-hand panels show coilin:mRFP; center panels, U2B″:GFP; right-hand panels, overlay. In all cells the CBs are greatly enlarged and in some cells are also more numerous than in the wild type. (A) Hypocotyl cell. (B) Leaf epidermal cell. (C) Root epidermal cell. (D) Root hair cell. (E) Trichome cell. Arrows show the presumed location of nucleoli. Bar, 5 μm.